Transformer-based spatial-temporal detection of apoptotic cell death in live-cell imaging.

Intravital microscopy has revolutionized live-cell imaging by allowing the study of spatial-temporal cell dynamics in living animals. However, the complexity of the data generated by this technology has limited the development of effective computational tools to identify and quantify cell processes. Amongst them, apoptosis is a crucial form of regulated cell death involved in tissue homeostasis and host defense. Live-cell imaging enabled the study of apoptosis at the cellular level, enhancing our understanding of its spatial-temporal regulation. However, at present, no computational method can deliver robust detection of apoptosis in microscopy timelapses. To overcome this limitation, we developed ADeS, a deep learning-based apoptosis detection system that employs the principle of activity recognition. We trained ADeS on extensive datasets containing more than 10,000 apoptotic instances collected both in vitro and in vivo, achieving a classification accuracy above 98% and outperforming state-of-the-art solutions. ADeS is the first method capable of detecting the location and duration of multiple apoptotic events in full microscopy timelapses, surpassing human performance in the same task. We demonstrated the effectiveness and robustness of ADeS across various imaging modalities, cell types, and staining techniques. Finally, we employed ADeS to quantify cell survival in vitro and tissue damage in mice, demonstrating its potential application in toxicity assays, treatment evaluation, and inflammatory dynamics. Our findings suggest that ADeS is a valuable tool for the accurate detection and quantification of apoptosis in live-cell imaging and, in particular, intravital microscopy data, providing insights into the complex spatial-temporal regulation of this process.

GPR182 is a broadly scavenging atypical chemokine receptor influencing T-independent immunity.

Immune responses highly depend on the effective trafficking of immune cells into and within secondary lymphoid organs (SLOs). Atypical chemokine receptors (ACKRs) scavenge chemokines to eliminate them from the extracellular space, thereby generating gradients that guide leukocytes. In contrast to canonical chemokine receptors, ACKRs do not induce classical intracellular signaling that results in cell migration. Recently, the closest relative of ACKR3, GPR182, has been partially deorphanized as a potential novel ACKR. We confirm and extend previous studies by identifying further ligands that classify GPR182 as a broadly scavenging chemokine receptor. We validate the "atypical" nature of the receptor, wherein canonical G-protein-dependent intracellular signaling is not activated following ligand stimulation. However, ß-arrestins are required for ligand-independent internalization and chemokine scavenging whereas the C-terminus is in part dispensable. In the absence of GPR182 in vivo, we observed elevated chemokine levels in the serum but also in SLO interstitium. We also reveal that CXCL13 and CCL28, which do not bind any other ACKR, are bound and efficiently scavenged by GPR182. Moreover, we found a cooperative relationship between GPR182 and ACKR3 in regulating serum CXCL12 levels, and between GPR182 and ACKR4 in controlling CCL20 levels. Furthermore, we unveil a new phenotype in GPR182-KO mice, in which we observed a reduced marginal zone (MZ), both in size and in cellularity, and thus in the T-independent antibody response. Taken together, we and others have unveiled a novel, broadly scavenging chemokine receptor, which we propose should be named ACKR5.

Tracking unlabeled cancer cells imaged with low resolution in wide migration chambers via U-NET class-1 probability (pseudofluorescence).

Cell migration is a pivotal biological process, whose dysregulation is found in many diseases including inflammation and cancer. Advances in microscopy technologies allow now to study cell migration in vitro, within engineered microenvironments that resemble in vivo conditions. However, to capture an entire 3D migration chamber for extended periods of time and with high temporal resolution, images are generally acquired with low resolution, which poses a challenge for data analysis. Indeed, cell detection and tracking are hampered due to the large pixel size (i.e., cell diameter down to 2 pixels), the possible low signal-to-noise ratio, and distortions in the cell shape due to changes in the z-axis position. Although fluorescent staining can be used to facilitate cell detection, it may alter cell behavior and it may suffer from fluorescence loss over time (photobleaching).Here we describe a protocol that employs an established deep learning method (U-NET), to specifically convert transmitted light (TL) signal from unlabeled cells imaged with low resolution to a fluorescent-like signal (class 1 probability). We demonstrate its application to study cancer cell migration, obtaining a significant improvement in tracking accuracy, while not suffering from photobleaching. This is reflected in the possibility of tracking cells for three-fold longer periods of time. To facilitate the application of the protocol we provide WID-U, an open-source plugin for FIJI and Imaris imaging software, the training dataset used in this paper, and the code to train the network for custom experimental settings.

ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production.

Chemotaxis is an essential physiological process, often harnessed by tumors for metastasis. CXCR4, its ligand CXCL12 and the atypical receptor ACKR3 are overexpressed in many human cancers. Interfering with this axis by ACKR3 deletion impairs lymphoma cell migration towards CXCL12. Here, we propose a model of how ACKR3 controls the migration of the diffused large B-cell lymphoma VAL cells in vitro and in vivo in response to CXCL12. VAL cells expressing full-length ACKR3, but not a truncated version missing the C-terminus, can support the migration of VAL cells lacking ACKR3 (VAL-ko) when allowed to migrate together. This migration of VAL-ko cells is pertussis toxin-sensitive suggesting the involvement of a Gi-protein coupled receptor. RNAseq analysis indicate the expression of chemotaxis-mediating LTB4 receptors in VAL cells. We found that LTB4 acts synergistically with CXCL12 in stimulating the migration of VAL cells. Pharmacologic or genetic inhibition of BLT1R markedly reduces chemotaxis towards CXCL12 suggesting that LTB4 enhances in a contact-independent manner the migration of lymphoma cells. The results unveil a novel mechanism of cell-to-cell-induced migration of lymphoma.

Marginal Zone Formation Requires ACKR3 Expression on B Cells.

The marginal zone (MZ) contributes to the highly organized spleen microarchitecture. We show that expression of atypical chemokine receptor 3 (ACKR3) defines two equal-sized populations of mouse MZ B cells (MZBs). ACKR3 is required for development of a functional MZ and for positioning of MZBs. Deletion of ACKR3 on B cells distorts the MZ, and MZBs fail to deliver antigens to follicles, reducing humoral responses. Reconstitution of MZ-deficient CD19ko mice shows that ACKR3- MZBs can differentiate into ACKR3+ MZBs, but not vice versa. The lack of a MZ is rescued by adoptive transfer of ACKR3-sufficient, and less by ACKR3-deficient, follicular B cells (FoBs); hence, ACKR3 expression is crucial for establishment of the MZ. The inability of CD19ko mice to respond to T-independent antigen is rescued when ACKR3-proficient, but not ACKR3-deficient, FoBs are transferred. Accordingly, ACKR3-deficient FoBs are able to reconstitute the MZ if the niche is pre-established by ACKR3-proficient MZBs.