Abstract
Cells collectively determine biological functions by communicating with each other—both through direct physical contact and secreted factors. Consequently, the local microenvironment of a ...cell influences its behavior, gene expression, and cellular crosstalk. Disruption of this microenvironment causes reciprocal changes in those features, which can lead to the development and progression of diseases. Hence, assessing the cellular transcriptome while simultaneously capturing the spatial relationships of cells within a tissue provides highly valuable insights into how cells communicate in health and disease. Yet, methods to probe the transcriptome often fail to preserve native spatial relationships, lack single-cell resolution, or are highly limited in throughput, i.e. lack the capacity to assess multiple environments simultaneously. Here, we introduce fragment-sequencing (fragment-seq), a method that enables the characterization of single-cell transcriptomes within multiple spatially distinct tissue microenvironments. We apply fragment-seq to a murine model of the metastatic liver to study liver zonation and the metastatic niche. This analysis reveals zonated genes and ligand-receptor interactions enriched in specific hepatic microenvironments. Finally, we apply fragment-seq to other tissues and species, demonstrating the adaptability of our method.
Sequencing-based spatial transcriptomics (ST) methods allow unbiased capturing of RNA molecules at barcoded spots, charting the distribution and localization of cell types and transcripts across a ...tissue. While the coarse resolution of these techniques is considered a disadvantage, we argue that the inherent proximity of transcriptomes captured on spots can be leveraged to reconstruct cellular networks. To this end, we developed ISCHIA (Identifying Spatial Co-occurrence in Healthy and InflAmed tissues), a computational framework to analyze the spatial co-occurrence of cell types and transcript species within spots. Co-occurrence analysis is complementary to differential gene expression, as it does not depend on the abundance of a given cell type or on the transcript expression levels, but rather on their spatial association in the tissue. We applied ISCHIA to analyze co-occurrence of cell types, ligands and receptors in a Visium dataset of human ulcerative colitis patients, and validated our findings at single-cell resolution on matched hybridization-based data. We uncover inflammation-induced cellular networks involving M cell and fibroblasts, as well as ligand-receptor interactions enriched in the inflamed human colon, and their associated gene signatures. Our results highlight the hypothesis-generating power and broad applicability of co-occurrence analysis on spatial transcriptomics data.
Synopsis
ISCHIA leverages the inherent proximity of spatial transcriptomics spot data to reconstruct networks of co-occurring cell types. Co-occurrence analysis is performed within and not across spots, and it is independent of cell type and transcript abundance.
ISCHIA applies co-occurrence analysis to spatial transcriptomics data to reconstruct cellular networks.
Co-occurrence analysis is complementary to differential gene expression, as it captures the spatial arrangement of cell types and transcripts in the tissue.
ISCHIA identifies an inflammation-induced cellular network involving M cells and fibroblasts in human ulcerative colitis samples.
ISCHIA leverages the inherent proximity of spatial transcriptomics spot data to reconstruct networks of co-occurring cell types. Co-occurrence analysis is performed within and not across spots, and is independent of cell type and transcript abundance.
Parenchymal migration of naive CD4
T cells in lymph nodes (LNs) is mediated by the Rac activator DOCK2 and PI3Kγ and is widely assumed to facilitate efficient screening of dendritic cells (DCs) ...presenting peptide-MHCs (pMHCs). Yet how CD4
T cell motility, DC density, and pMHC levels interdependently regulate such interactions has not been comprehensively examined. Using intravital imaging of reactive LNs in DC-immunized mice, we show that pMHC levels determined the occurrence and timing of stable CD4
T cell-DC interactions. Despite the variability in interaction parameters, ensuing CD4
T cell proliferation was comparable over a wide range of pMHC levels. Unexpectedly, decreased intrinsic motility of DOCK2
CD4
T cells did not impair encounters with DCs in dense paracortical networks and, instead, increased interaction stability, whereas PI3Kγ deficiency had no effect on interaction parameters. In contrast, intravital and whole-organ imaging showed that DOCK2-driven T cell motility was required to detach from pMHC
DCs and to find rare pMHC
DCs. In sum, our data uncover flexible signal integration by scanning CD4
T cells, suggesting a search strategy evolved to detect low-frequency DCs presenting high cognate pMHC levels.
T cells are actively scanning pMHC-presenting cells in lymphoid organs and nonlymphoid tissues (NLTs) with divergent topologies and confinement. How the T cell actomyosin cytoskeleton facilitates ...this task in distinct environments is incompletely understood. Here, we show that lack of Myosin IXb (Myo9b), a negative regulator of the small GTPase Rho, led to increased Rho-GTP levels and cell surface stiffness in primary T cells. Nonetheless, intravital imaging revealed robust motility of Myo9b
CD8
T cells in lymphoid tissue and similar expansion and differentiation during immune responses. In contrast, accumulation of Myo9b
CD8
T cells in NLTs was strongly impaired. Specifically, Myo9b was required for T cell crossing of basement membranes, such as those which are present between dermis and epidermis. As consequence, Myo9b
CD8
T cells showed impaired control of skin infections. In sum, we show that Myo9b is critical for the CD8
T cell adaptation from lymphoid to NLT surveillance and the establishment of protective tissue-resident T cell populations.
T cell activation in lymphoid tissue occurs through interactions with cognate peptide-major histocompatibility complex (pMHC)-presenting dendritic cells (DCs). Intravital imaging studies using
...peptide-pulsed DCs have uncovered that cognate pMHC levels imprint a wide range of dynamic contacts between these two cell types. T cell-DC interactions vary between transient, "kinapse-like" contacts at low to moderate pMHC levels to immediate "synapse-like" arrest at DCs displaying high pMHC levels. To date, it remains unclear whether this pattern is recapitulated when the immune system faces a replicative agent, such as a virus, at low and high inoculum. Here, we locally administered low and high inoculum of lymphocytic choriomeningitis virus (LCMV) in mice to follow activation parameters of Ag-specific CD4
and CD8
T cells in draining lymph nodes (LNs) during the first 72 h post infection. We correlated these data with kinapse- and synapse-like motility patterns of Ag-specific T cells obtained by intravital imaging of draining LNs. Our data show that initial viral inoculum controls immediate synapse-like T cell arrest vs. continuous kinapse-like motility. This remains the case when the viral inoculum and thus the inflammatory microenvironment in draining LNs remains identical but cognate pMHC levels vary. Our data imply that the Ag-processing capacity of draining LNs is equipped to rapidly present high levels of cognate pMHC when antigenic material is abundant. Our findings further suggest that widespread T cell arrest during the first 72 h of an antimicrobial immune responses is not required to trigger proliferation. In sum, T cells adapt their scanning behavior according to available antigen levels during viral infections, with dynamic changes in motility occurring before detectable expression of early activation markers.
Here, we provide detailed protocols for the isolation of mouse Kupffer cells – the liver-resident macrophages – for phenotypic (e.g., via flow cytometry, mass cytometry, or RNA-sequencing) analyses ...or for functional experiments involving cell culture. The procedures presented can be adapted for the isolation of other hepatic cell populations.
For complete details on the use and execution of this protocol, please refer to De Simone et al. (2021).
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•Protocol for Kupffer cell (KC) isolation•Suitable for the simultaneous isolation of other hepatic cell populations•Isolated KCs are suitable for phenotypic and functional analyses•We provide critical tips for cell processing and FACS-based sorting
Here, we provide detailed protocols for the isolation of mouse Kupffer cells – the liver-resident macrophages – for phenotypic (e.g., via flow cytometry, mass cytometry or RNA-sequencing) analyses or for functional experiments involving cell culture. The procedures presented can be adapted for the isolation of other hepatic cell populations.
New World arenaviruses are rodent-transmitted viruses and include a number of pathogens that are responsible for causing severe human disease. This includes Junín virus (JUNV), which is the causative ...agent of Argentine hemorrhagic fever. The wild nature and mobility of the rodent reservoir host makes it difficult to control the disease, and currently passive immunization with high-titer neutralizing antibody-containing plasma from convalescent patients is the only specific therapy. However, dwindling supplies of naturally available convalescent plasma, and challenges in developing similar resources for other closely related viruses, have made the development of alternative antibody-based therapeutic approaches of critical importance. In this study, we sought to induce a neutralizing antibody response in rabbits against the receptor-binding subunit of the viral glycoprotein, GP1, and the specific peptide sequences in GP1 involved in cellular receptor contacts. While these specific receptor-interacting peptides did not efficiently induce the production of neutralizing antibodies when delivered as a particulate antigen (as part of hepatitis B virus core-like particles), we showed that recombinant JUNV GP1 purified from transfected mammalian cells induced virus-neutralizing antibodies at high titers in rabbits. Further, neutralization was observed across a range of unrelated JUNV strains, a feature that is critical for effectiveness in the field. These results underscore the potential of GP1 alone to induce a potent neutralizing antibody response and highlight the importance of epitope presentation. In addition, effective virus neutralization by rabbit antibodies supports the potential applicability of this species for the future development of immunotherapeutics (e.g., based on humanized monoclonal antibodies). Such information can be applied in the design of vaccines and immunogens for both prevention and specific therapies against this and likely also other closely related pathogenic New World arenaviruses.
The liver is the target of several infectious, inflammatory, and neoplastic diseases, which affect hundreds of millions of people worldwide and cause an estimated death toll of more than 2 million ...people each year. Dysregulation of T cell responses has been implicated in the pathogenesis of these diseases; hence, it is critically important to understand the function and fate of T cells in the liver. Here, we provide an overview of the current knowledge on liver immune surveillance by conventional and invariant T cells and explore the complex cross-talk between immune cell subsets that determines the balance between hepatic immunity and tolerance.
The serine and threonine kinase MST1 is the mammalian homolog of Hippo. MST1 is a critical mediator of the migration, adhesion, and survival of T cells; however, these functions of MST1 are ...independent of signaling by its typical effectors, the kinase LATS and the transcriptional coactivator YAP. The kinase NDR1, a member of the same family of kinases as LATS, functions as a tumor suppressor by preventing T cell lymphomagenesis, which suggests that it may play a role in T cell homeostasis. We generated and characterized mice with a T cell-specific double knockout of Ndr1 and Ndr2 (Ndr DKO). Compared with control mice, Ndr DKO mice exhibited a substantial reduction in the number of naïve T cells in their secondary lymphoid organs. Mature single-positive thymocytes accumulated in the thymus in Ndr DKO mice. We also found that NDRs acted downstream of MST1 to mediate the egress of mature thymocytes from the thymus, as well as the interstitial migration of naïve T cells within popliteal lymph nodes. Together, our findings indicate that the kinases NDR1 and NDR2 function as downstream effectors of MST1 to mediate thymocyte egress and T cell migration.
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