Tumor-activated lymph node fibroblasts suppress T cell function in diffuse large B cell lymphoma.

Recent transcriptomic-based analysis of diffuse large B cell lymphoma (DLBCL) has highlighted the clinical relevance of LN fibroblast and tumor-infiltrating lymphocyte (TIL) signatures within the tumor microenvironment (TME). However, the immunomodulatory role of fibroblasts in lymphoma remains unclear. Here, by studying human and mouse DLBCL-LNs, we identified the presence of an aberrantly remodeled fibroblastic reticular cell (FRC) network expressing elevated fibroblast-activated protein (FAP). RNA-Seq analyses revealed that exposure to DLBCL reprogrammed key immunoregulatory pathways in FRCs, including a switch from homeostatic to inflammatory chemokine expression and elevated antigen-presentation molecules. Functional assays showed that DLBCL-activated FRCs (DLBCL-FRCs) hindered optimal TIL and chimeric antigen receptor (CAR) T cell migration. Moreover, DLBCL-FRCs inhibited CD8+ TIL cytotoxicity in an antigen-specific manner. Notably, the interrogation of patient LNs with imaging mass cytometry identified distinct environments differing in their CD8+ TIL-FRC composition and spatial organization that associated with survival outcomes. We further demonstrated the potential to target inhibitory FRCs to rejuvenate interacting TILs. Cotreating organotypic cultures with FAP-targeted immunostimulatory drugs and a bispecific antibody (glofitamab) augmented antilymphoma TIL cytotoxicity. Our study reveals an immunosuppressive role of FRCs in DLBCL, with implications for immune evasion, disease pathogenesis, and optimizing immunotherapy for patients.

Disrupted Peyer's Patch Microanatomy in COVID-19 Including Germinal Centre Atrophy Independent of Local Virus.

Confirmed SARS-coronavirus-2 infection with gastrointestinal symptoms and changes in microbiota associated with coronavirus disease 2019 (COVID-19) severity have been previously reported, but the disease impact on the architecture and cellularity of ileal Peyer's patches (PP) remains unknown. Here we analysed post-mortem tissues from throughout the gastrointestinal (GI) tract of patients who died with COVID-19. When virus was detected by PCR in the GI tract, immunohistochemistry identified virus in epithelium and lamina propria macrophages, but not in lymphoid tissues. Immunohistochemistry and imaging mass cytometry (IMC) analysis of ileal PP revealed depletion of germinal centres (GC), disruption of B cell/T cell zonation and decreased potential B and T cell interaction and lower nuclear density in COVID-19 patients. This occurred independent of the local viral levels. The changes in PP demonstrate that the ability to mount an intestinal immune response is compromised in severe COVID-19, which could contribute to observed dysbiosis.

High-Dimensional Single-Cell Quantitative Profiling of Skeletal Muscle Cell Population Dynamics during Regeneration.

The interstitial space surrounding the skeletal muscle fibers is populated by a variety of mononuclear cell types. Upon acute or chronic insult, these cell populations become activated and initiate finely-orchestrated crosstalk that promotes myofiber repair and regeneration. Mass cytometry is a powerful and highly multiplexed technique for profiling single-cells. Herein, it was used to dissect the dynamics of cell populations in the skeletal muscle in physiological and pathological conditions. Here, we characterized an antibody panel that could be used to identify most of the cell populations in the muscle interstitial space. By exploiting the mass cytometry resolution, we provided a comprehensive picture of the dynamics of the major cell populations that sensed and responded to acute damage in wild type mice and in a mouse model of Duchenne muscular dystrophy. In addition, we revealed the intrinsic heterogeneity of many of these cell populations.

IGHV sequencing reveals acquired N-glycosylation sites as a clonal and stable event during follicular lymphoma evolution.

Follicular lymphoma B cells undergo continuous somatic hypermutation (SHM) of their immunoglobulin variable region genes, generating a heterogeneous tumor population. SHM introduces DNA sequences encoding N-glycosylation sites asparagine-X-serine/threonine (N-gly sites) within the V-region that are rarely found in normal B-cell counterparts. Unique attached oligomannoses activate B-cell receptor signaling pathways after engagement with calcium-dependent lectins expressed by tissue macrophages. This novel interaction appears critical for tumor growth and survival. To elucidate the significance of N-gly site presence and loss during ongoing SHM, we tracked site behavior during tumor evolution and progression in a diverse group of patients through next-generation sequencing. A hierarchy of subclones was visualized through lineage trees based on SHM semblance between subclones and their discordance from the germline sequence. We observed conservation of N-gly sites in more than 96% of subclone populations within and across diagnostic, progression, and transformation events. Rare N-gly-negative subclones were lost or negligible from successive events, in contrast to N-gly-positive subclones, which could additionally migrate between anatomical sites. Ongoing SHM of the N-gly sites resulted in subclones with different amino acid compositions across disease events, yet the vast majority of resulting DNA sequences still encoded for an N-gly site. The selection and expansion of only N-gly-positive subclones is evidence of the tumor cells' dependence on sites, despite the changing genomic complexity as the disease progresses. N-gly sites were gained in the earliest identified lymphoma cells, indicating they are an early and stable event of pathogenesis. Targeting the inferred mannose-lectin interaction holds therapeutic promise.

HiPPO and PANDA: Two Bioinformatics Tools to Support Analysis of Mass Cytometry Data.

High-dimensional mass cytometry (Cytometry by Time-Of-Flight; CyTOF) is a multiparametric single-cell approach that allows for more than 40 parameters to be evaluated simultaneously, opening the possibility to dissect cellular heterogeneity and elucidate functional interactions between different cell types. However, the complexity of these data makes analysis and interpretation daunting. We created High-throughput Population Profiler (HiPPO), a tool that reduces the complexity of the CyTOF data and allows homogeneous clusters of cells to be visualized in an intuitive manner. Each subpopulation is mapped to the Population Analysis Database (PANDA), an open-source, manually curated database containing protein expression profiles for selected markers of primary cells, allowing for cell type abundance in the analyzed samples to be monitored. Custom cell definitions can be submitted for targeted identifications. All cell clusters, regardless of their annotation status, are available for further analyses. HiPPO also conducts nonparametric tests to determine whether differences in protein expression levels between conditions are significant. HiPPO strikes a balance between diagnostic power and computational burden. Its minimal computational footprint allows for subpopulations in a heterogeneous sample to be identified and quantified quickly.

Fibro-adipogenic progenitors of dystrophic mice are insensitive to NOTCH regulation of adipogenesis.

Fibro-adipogenic progenitors (FAPs) promote satellite cell differentiation in adult skeletal muscle regeneration. However, in pathological conditions, FAPs are responsible for fibrosis and fatty infiltrations. Here we show that the NOTCH pathway negatively modulates FAP differentiation both in vitro and in vivo. However, FAPs isolated from young dystrophin-deficient mdx mice are insensitive to this control mechanism. An unbiased mass spectrometry-based proteomic analysis of FAPs from muscles of wild-type and mdx mice suggested that the synergistic cooperation between NOTCH and inflammatory signals controls FAP differentiation. Remarkably, we demonstrated that factors released by hematopoietic cells restore the sensitivity to NOTCH adipogenic inhibition in mdx FAPs. These results offer a basis for rationalizing pathological ectopic fat infiltrations in skeletal muscle and may suggest new therapeutic strategies to mitigate the detrimental effects of fat depositions in muscles of dystrophic patients.

The immunosuppressant drug azathioprine restrains adipogenesis of muscle Fibro/Adipogenic Progenitors from dystrophic mice by affecting AKT signaling.

Fibro/Adipogenic Progenitors (FAPs) define a stem cell population playing a pro-regenerative role after muscle damage. When removed from their natural niche, FAPs readily differentiate into adipocytes or fibroblasts. This digressive differentiation potential, which is kept under tight control in the healthy muscle niche, contributes to fat and scar infiltrations in degenerative myopathies, such as in Duchenne Muscular Dystrophy (DMD). Controlling FAP differentiation by means of small molecules may contribute to delay the adverse consequences of the progressive pathological degeneration while offering, at the same time, a wider temporal window for gene therapy and cell-based strategies. In a high content phenotypic screening, we identified the immunosuppressant, azathioprine (AZA) as a negative modulator of FAP adipogenesis. We show here that AZA negatively affects the adipogenic propensity of FAPs purified from wild type and mdx mice by impairing the expression of the master adipogenic regulator, peroxisome proliferator-activated receptor γ (PPARγ). We show that this inhibition correlates with a decline in the activation of the AKT-mTOR axis, the main pathway that transduces the pro-adipogenic stimulus triggered by insulin. In addition, AZA exerts a cytostatic effect that has a negative impact on the mitotic clonal process that is required for the terminal differentiation of the preadipocyte-committed cells.

Single-cell mass cytometry and transcriptome profiling reveal the impact of graphene on human immune cells.

Understanding the biomolecular interactions between graphene and human immune cells is a prerequisite for its utilization as a diagnostic or therapeutic tool. To characterize the complex interactions between graphene and immune cells, we propose an integrative analytical pipeline encompassing the evaluation of molecular and cellular parameters. Herein, we use single-cell mass cytometry to dissect the effects of graphene oxide (GO) and GO functionalized with amino groups (GONH2) on 15 immune cell populations, interrogating 30 markers at the single-cell level. Next, the integration of single-cell mass cytometry with genome-wide transcriptome analysis shows that the amine groups reduce the perturbations caused by GO on cell metabolism and increase biocompatibility. Moreover, GONH2 polarizes T-cell and monocyte activation toward a T helper-1/M1 immune response. This study describes an innovative approach for the analysis of the effects of nanomaterials on distinct immune cells, laying the foundation for the incorporation of single-cell mass cytometry on the experimental pipeline.

Opposing roles of Nfkb2 gene products p100 and p52 in the regulation of breast cancer stem cells.

PURPOSE: Nuclear factor-kappa B (NF-κB) signalling has been shown to regulate properties of breast cancer stem cells. However, the specific contribution of the non-canonical NF-κB pathway, components of which are elevated in aggressive breast cancer has not been addressed. METHODS: Through shRNA silencing of the Nfkb2 gene, the role of p100/p52 in 4T1 and N202.1A cell lines were assessed by NF-κB reporter, invasion, tumoursphere and orthotopic transplantation assays. The processing of p100 into p52 was also inhibited with a p97 ATPase inhibitor, NMS-873, and its effects on tumoursphere formation was assessed. RESULTS: Knockdown of Nfkb2 led to opposing changes in NF-κB-dependent transcription. NF-κB activity was elevated in 4T1 cells and this resulted in increased motility, cancer stem cell (CSC) activity and tumourigenicity in vivo. Conversely, depletion of Nfkb2 in N202.1a cells decreased NF-κB activity, CSC properties and tumourigenicity in vivo. By selectively overexpressing the p52 subunit in Nfkb2 depleted cells, we found that the increased malignancy in 4T1 cells could not be reverted in the presence of p52, whereas the decreased tumourigenicity of N202.1a cells could be rescued by p52. These results indicate that p100 and its subunit p52 have opposing effects on breast CSC activity. Accordingly, inhibition of an upstream regulator of p100 processing was effective in reducing tumoursphere formation of N202.1A and SKBR3 (ErbB2 HIGH) cells without aggravating that of 4T1 and MDA-MB-231 (ErbB2LOW) cells. CONCLUSION: These findings indicate that inhibiting the processing of p100 may be a potential therapeutic strategy to suppress CSC activity in a subset of breast tumours.

Characterization by mass cytometry of different methods for the preparation of muscle mononuclear cells.

Biological processes that are mediated by cell-cell interactions in heterogeneous populations are best approached by methods that have single cell resolution. Most of these methods rely on the preparation, from solid tissues, of cell suspensions by enzymatic digestion, followed by analysis of single cell reactivity to an antibody panel that allows the discrimination of cell populations and characterization of their activation state. Thus for any specific biological problem, both efficient and at the same time mild, protocols for cell separation, together with tissue specific panels of antibodies, need to be developed and optimized. Here we characterize an antibody panel that permits the discrimination of mononuclear muscle cell populations by mass cytometry and use it to characterize the cell populations obtained by three different cell extraction procedures from muscle fibers. We show that our panel of antibodies, albeit limited and incomplete, is sufficient to discriminate most of the mononuclear muscle cell populations and that each cell extraction method yields heterogeneous cell populations with a different relative abundance of the distinct cell types.

SIGNOR: a database of causal relationships between biological entities.

Assembly of large biochemical networks can be achieved by confronting new cell-specific experimental data with an interaction subspace constrained by prior literature evidence. The SIGnaling Network Open Resource, SIGNOR (available on line at http://signor.uniroma2.it), was developed to support such a strategy by providing a scaffold of prior experimental evidence of causal relationships between biological entities. The core of SIGNOR is a collection of approximately 12,000 manually-annotated causal relationships between over 2800 human proteins participating in signal transduction. Other entities annotated in SIGNOR are complexes, chemicals, phenotypes and stimuli. The information captured in SIGNOR can be represented as a signed directed graph illustrating the activation/inactivation relationships between signalling entities. Each entry is associated to the post-translational modifications that cause the activation/inactivation of the target proteins. More than 4900 modified residues causing a change in protein concentration or activity have been curated and linked to the modifying enzymes (about 351 human kinases and 94 phosphatases). Additional modifications such as ubiquitinations, sumoylations, acetylations and their effect on the modified target proteins are also annotated. This wealth of structured information can support experimental approaches based on multi-parametric analysis of cell systems after physiological or pathological perturbations and to assemble large logic models.