Butyrophilin-like (Btnl) genes are emerging as major epithelial determinants of tissue-associated γδ T cell compartments. Thus, the development of signature, murine TCRγδ⁺ intraepithelial lymphocytes (IEL) in gut and skin depends on Btnl family members, Btnl1 and Skint1, respectively. In seeking mechanisms underlying these profound effects, we now show that normal gut and skin γδ IEL development additionally requires Btnl6 and Skint2, respectively, and furthermore that different Btnl heteromers can seemingly shape different intestinal γδ⁺ IEL repertoires. This formal genetic evidence for the importance of Btnl heteromers also applied to the steady-state, since sustained Btnl expression is required to maintain the signature TCR.Vγ7⁺ IEL phenotype, including specific responsiveness to Btnl proteins. In sum, Btnl proteins are required to select and to maintain the phenotypes of tissue-protective γδ IEL compartments, with combinatorially diverse heteromers having differential impacts on different IEL subsets.

A dense population of embryo-derived Langerhans cells (eLCs) is maintained within the sealed epidermis without contribution from circulating cells. When this network is perturbed by transient exposure to ultraviolet light, short-term LCs are temporarily reconstituted from an initial wave of monocytes but thought to be superseded by more permanent repopulation with undefined LC precursors. However, the extent to which this process is relevant to immunopathological processes that damage LC population integrity is not known. Using a model of allogeneic hematopoietic stem cell transplantation, where alloreactive T cells directly target eLCs, we have asked whether and how the original LC network is ultimately restored. We find that donor monocytes, but not dendritic cells, are the precursors of long-term LCs in this context. Destruction of eLCs leads to recruitment of a wave of monocytes that engraft in the epidermis and undergo a sequential pathway of differentiation via transcriptionally distinct EpCAM⁺ precursors. Monocyte-derived LCs acquire the capacity of self-renewal, and proliferation in the epidermis matched that of steady-state eLCs. However, we identified a bottleneck in the differentiation and survival of epidermal monocytes, which, together with the slow rate of renewal of mature LCs, limits repair of the network. Furthermore, replenishment of the LC network leads to constitutive entry of cells into the epidermal compartment. Thus, immune injury triggers functional adaptation of mechanisms used to maintain tissue-resident macrophages at other sites, but this process is highly inefficient in the skin.

Viral RNA synthesis of several non-segmented, negative-sense RNA viruses (NNSVs) takes place in inclusion bodies (IBs) that show properties of liquid organelles, which are formed by liquid-liquid phase separation of scaffold proteins. It is believed that this is driven by intrinsically disordered regions (IDRs) and/or multiple copies of interaction domains, which for NNSVs are usually located in their nucleo - and phosphoproteins. In contrast to other NNSVs, the Ebola virus (EBOV) nucleoprotein NP alone is sufficient to form IBs without the need for a phosphoprotein, and to facilitate the recruitment of other viral proteins into these structures. While it has been proposed that also EBOV IBs are liquid organelles, this has so far not been formally demonstrated. Here we used a combination of live cell microscopy, fluorescence recovery after photobleaching assays, and mutagenesis approaches together with reverse genetics-based generation of recombinant viruses to study the formation of EBOV IBs. Our results demonstrate that EBOV IBs are indeed liquid organelles, and that oligomerization but not IDRs of the EBOV nucleoprotein plays a key role in their formation. Additionally, VP35 (often considered the phosphoprotein-equivalent of EBOV) is not essential for IB formation, but alters their liquid behaviour. These findings define the molecular mechanism for the formation of EBOV IBs, which play a central role in the life cycle of this deadly virus.

Filoviruses such as the Ebola virus (EBOV) cause severe hemorrhagic fevers with high case fatality rates and limited treatment options. The identification of virus-host cell interactions shared among several viruses would represent promising targets for the development of broadly active antivirals.

Lung immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for the ability to deal with infection. Using light sheet fluorescence microscopy of hamster lung slices in combination with virological, immunohistochemical, and RNA sequencing analyses, we show a wave of monocyte-derived macrophage (MDM) infiltration and the antiviral response which follows the spread of SARS-CoV-2 through the lungs and lead to virus elimination. These innate immune processes are related to the onset of necrotizing inflammatory and remodeling responses, which manifest as extensive cell death, vascular damage, and cell proliferation. We show that MDM appear in parallel with virus clearance and endothelial injury. Prothrombotic factor upregulation, tissue repair, and alveolar cell proliferation result in tissue remodeling, which is followed by fibrosis despite a decrease in inflammatory and antiviral activities. Although the lung tissue integrity is repaired, longer-term alterations of the lung arise as an outcome of concurrent tissue damage and regeneration processes. IMPORTANCE: We present the first study of the 3D kinetics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the early host response in a large lung volume using a combination of tissue imaging and transcriptomics. This approach allowed us to make a number of important findings: Spatially restricted antiviral response is shown, including the formation of monocytic macrophage clusters and upregulation of the major histocompatibility complex II in infected epithelial cells. The monocyte-derived macrophages are linked to SARS-CoV-2 clearance, and the appearance of these cells is associated with post-infection endothelial damage; thus, we shed light on the role of these cells in infected tissue. An early onset of tissue repair occurring simultaneously with inflammatory and necrotizing processes provides the basis for longer-term alterations in the lungs.

We applied fluorescence lifetime imaging microscopy to map the microenvironment of the myosin essential light chain (ELC) in permeabilized skeletal muscle fibers. Four ELC mutants containing a single cysteine residue at different positions in the C-terminal half of the protein (ELC-127, ELC-142, ELC-160, and ELC-180) were generated by site-directed mutagenesis, labeled with 7-diethylamino-3-((((2-iodoacetamido)ethyl)amino)carbonyl)coumarin, and introduced into permeabilized rabbit psoas fibers. Binding to the myosin heavy chain was associated with a large conformational change in the ELC. When the fibers were moved from relaxation to rigor, the fluorescence lifetime increased for all label positions. However, when 1% stretch was applied to the rigor fibers, the lifetime decreased for ELC-127 and ELC-180 but did not change for ELC-142 and ELC-160. The differential change of fluorescence lifetime demonstrates the shift in position of the C-terminal domain of ELC with respect to the heavy chain and reveals specific locations in the lever arm region sensitive to the mechanical strain propagating from the actin-binding site to the lever arm.