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Additional file 1. Fig S1. Metacells are compatible with UMAP visualization. Fig S2. Metacell size and gene coverage distributions. Fig S3. Metacells recover rare cell types. Fig S4. Metacells preserve clustering. Fig S5. Metacells are compatible with unweighted clustering. Fig S6. Clustering of metacells is robust to different ways of building metacells. Fig S7. Metacells are compatible with unweighted differential expression analysis. Fig S8. Differential expression of metacells is robust to different ways of building metacells. Fig S9. Metacells preserve differential expression between conditions. Fig S10. Gating strategy for the flow cytometry analysis of DCs from murine KP1.9 lung adenocarcinoma for one representative sample. Fig S11. Cell type annotation in the Cd8_TILs dataset. Fig S12. Conservation of RNA velocity results in the brain_cells dataset. Fig S13. Conservation of RNA velocity results in the pancreatic_cells dataset. Fig S14. Computational time and memory allocation for RNA velocity. Fig S15. Approximate coarse-graining in SuperCell. Fig S16. Computational time and memory allocation for metacell construction and downstream analyses. Table S1. Datasets used for the analysis. Table S2. Datasets integrated in the TIM_atlas dataset. Table S3. Genes ranked better in differential expression analysis (cDC vs pDC) at metacell level. Table S4. Genes ranked better in differential expression analysis (pDC vs cDC) at metacell level. Table S5. Antibodies used in flow cytometry (Fig. 2f, Additional file 1: Fig. S11).

Immunotherapy is revolutionizing cancer treatment but is often restricted by toxicities. What distinguishes adverse events from concomitant antitumor reactions is poorly understood. Here, using anti-CD40-treatment in mice as a model of Th1-promoting immunotherapy, we showed that liver macrophages promoted local immune-related adverse events. Mechanistically, tissue-resident Kupffer cells mediated liver toxicity by sensing lymphocyte-derived IFN-𝛾 and subsequently producing IL-12. Conversely, dendritic cells were dispensable for toxicity but drove tumor control. IL-12 and IFN-𝛾 were not toxic themselves, but prompted a neutrophil response that determined the severity of tissue damage. We observed activation of similar inflammatory pathways following anti-PD-1 and anti-CTLA4 immunotherapies in mice and humans. These findings implicated macrophages and neutrophils as mediators and effectors of aberrant inflammation in Th1-promoting immunotherapy, suggesting distinct mechanisms of toxicity and antitumor immunity.

Single-cell RNA sequencing (scRNA-seq) technologies offer unique opportunities for exploring heterogeneous cell populations. However, in-depth single-cell transcriptomic characterization of complex tissues often requires profiling tens to hundreds of thousands of cells. Such large numbers of cells represent an important hurdle for downstream analyses, interpretation and visualization. Here we develop a network-based coarse-graining framework where highly similar cells are merged into super-cells. We demonstrate that super-cells not only preserve but often improve the results of downstream analyses including visualization, clustering, differential expression, cell type annotation, gene correlation, imputation, RNA velocity and data integration. By capitalizing on the redundancy inherent to scRNA-seq data, super-cells significantly facilitate and accelerate the construction and interpretation of single-cell atlases, as demonstrated by the integration of 1.46 million cells from COVID-19 patients in less than two hours on a standard desktop.

Cytotoxic T lymphocyte (CTL) responses against tumors are maintained by stem-like memory cells that self-renew but also give rise to effector-like cells. The latter gradually lose their anti-tumor activity and acquire an epigenetically fixed, hypofunctional state, leading to tumor tolerance. Here, we show that the conversion of stem-like into effector-like CTLs involves a major chemotactic reprogramming that includes the upregulation of chemokine receptor CXCR6. This receptor positions effector-like CTLs in a discrete perivascular niche of the tumor stroma that is densely occupied by CCR7+ dendritic cells (DCs) expressing the CXCR6 ligand CXCL16. CCR7+ DCs also express and trans-present the survival cytokine interleukin-15 (IL-15). CXCR6 expression and IL-15 trans-presentation are critical for the survival and local expansion of effector-like CTLs in the tumor microenvironment to maximize their anti-tumor activity before progressing to irreversible dysfunction. These observations reveal a cellular and molecular checkpoint that determines the magnitude and outcome of anti-tumor immune responses.

Background Immune checkpoint blockade (ICB) with antibodies against PD-1 or PD-L1 may provide therapeutic benefits in patients with non-small cell lung cancer (NSCLC). However, most tumours are resistant and cases of disease hyper-progression have also been reported. Materials and Methods Genetically engineered mouse models of KrasG12Dp53null NSCLC were treated with cisplatin along with antibodies against angiopoietin-2/VEGFA, PD-1 and CSF1R. Tumour growth was monitored by micro-computed tomography and the tumour vasculature and immune cell infiltrates were assessed by immunofluorescence staining and flow cytometry. Results Combined angiopoietin-2/VEGFA blockade by a bispecific antibody (A2V) modulated the vasculature and abated immunosuppressive macrophages while increasing CD8+effector T cells in the tumours, achieving disease stabilization comparable or superior to cisplatin-based chemotherapy. However, these immunological responses were unexpectedly limited by the addition of a PD-1 antibody, which paradoxically enhanced progression of a fraction of the tumours through a mechanism involving regulatory T cells and macrophages. Elimination of tumour-associated macrophages with a CSF1R-blocking antibody induced NSCLC regression in combination with PD-1 blockade and cisplatin. Conclusions The immune cell composition of the tumour determines the outcome of PD-1 blockade. In NSCLC, high infiltration of regulatory T cells and immunosuppressive macrophages may account for tumour hyper-progression upon ICB. Disclosure Information A. Martinez-Usatorre: None. E. Kadioglu: None. C. Cianciaruso: None. B. Torchia: None. J. Faget: None. E. Meylan: None. M. Schmittnaegel: None. I. Keklikoglou: None. M. De Palma: None.

Immune checkpoint blockade (ICB) with PD-1 or PD-L1 antibodies has been approved for the treatment of non-small cell lung cancer (NSCLC). However, only a minority of patients respond, and sustained remissions are rare. Both chemotherapy and anti-angiogenic drugs may improve the efficacy of ICB in mouse tumor models and patients with cancer. Here, we used genetically engineered mouse models of Kras (G12D/+);p53 (-/-) NSCLC, including a mismatch repair-deficient variant (Kras (G12D/+);p53 (-/-);Msh2 (-/-)) with higher mutational burden, and longitudinal imaging to study tumor response and resistance to combinations of ICB, anti-angiogenic therapy, and chemotherapy. Anti-angiogenic blockade of vascular endothelial growth factor A (VEGFA) and angiopoietin-2 (ANGPT2) markedly slowed progression of autochthonous lung tumors but, contrary to findings in other cancer types, addition of a PD-1 or PD-L1 antibody was not beneficial and even accelerated progression of a fraction of the tumors. We found that anti-angiogenic treatment facilitated tumor infiltration by PD-1(+) regulatory T cells (T(regs)), which were more efficiently targeted by the PD-1 antibody than CD8(+) T cells. Both tumor-associated macrophages (TAMs) of monocyte origin, which are colony-stimulating factor 1 receptor (CSF1R)-dependent, and TAMs of alveolar origin, which are sensitive to cisplatin, contributed to establish a transforming growth factor-β (TGFβ)-rich tumor microenvironment that supported PD-1(+) T(regs). Dual TAM targeting with a combination of a CSF1R inhibitor and cisplatin abated T(regs), redirected the PD-1 antibody to CD8(+) T cells, and improved the efficacy of anti-angiogenic immunotherapy, achieving regression of most tumors.

Biosensors are indispensable tools for public, global, and personalized healthcare as they provide tests that can be used from early disease detection and treatment monitoring to preventing pandemics. We introduce single-wavelength imaging biosensors capable of reconstructing spectral shift information induced by biomarkers dynamically using an advanced data processing technique based on an optimal linear estimator. Our method achieves superior sensitivity without wavelength scanning or spectroscopy instruments. We engineered diatomic dielectric metasurfaces supporting bound states in the continuum that allows high-quality resonances with accessible near-fields by in-plane symmetry breaking. The large-area metasurface chips are configured as microarrays and integrated with microfluidics on an imaging platform for real-time detection of breast cancer extracellular vesicles encompassing exosomes. The optofluidic system has high sensing performance with nearly 70 1/RIU figure-of-merit enabling detection of on average 0.41 nanoparticle/µm2 and real-time measurements of extracellular vesicles binding from down to 204 femtomolar solutions. Our biosensors provide the robustness of spectrometric approaches while substituting complex instrumentation with a single-wavelength light source and a complementary-metal-oxide-semiconductor camera, paving the way toward miniaturized devices for point-of-care diagnostics., The authors engineer a type of bound states in the continuum in diatomic dielectric metasurfaces, allowing for high-quality resonances with accessible enhanced fields. Metasurface microarrays are integrated with microfluidics on an imaging platform for real-time detection of biosamples, based on reconstructing spectral shift information.

Pancreatic beta cells synthesize and secrete the neurotransmitter GABA (gamma-aminobutyric acid) as a paracrine and autocrine signal to help regulate hormone secretion and islet homeostasis. Islet GABA release has classically been described as a secretory-vesicle-mediated event. Yet, a limitation of the hypothesized vesicular GABA release from islets is the lack of expression of a vesicular GABA transporter in beta cells. Consequentially, GABA accumulates in the cytosol. Here, we provide evidence that the human beta cell effluxes GABA from a cytosolic pool in a pulsatile manner, imposing a synchronizing rhythm on pulsatile insulin secretion. The volume regulatory anion channel, functionally encoded by LRRC8A or Swell1, is critical for pulsatile GABA secretion. GABA content in beta cells is depleted and secretion is disrupted in islets from patients with type 1 and type 2 diabetes, suggesting that loss of GABA as a synchronizing signal for hormone output may correlate with diabetes pathogenesis.

Immune checkpoint blockade (ICB) with PD-1 or PD-L1 antibodies has been approved for the treatment of non-small cell lung cancer (NSCLC), often in combination with chemotherapy. However, only a minority of the patients respond and sustained remissions are rare. Anti-angiogenic therapy improves tumor response to ICB in mouse models of cancer and in patients with EGFR-mutant NSCLC, but the contribution of anti-angiogenic therapy to the clinical efficacy of ICB in patients with KRAS-mutant NSCLC remains unclear. We used genetically engineered mouse models of KrasG12D/p53null NSCLC, including a mismatch repair-deficient variant (KrasG12D/p53null/Msh2null) with higher mutational burden, and longitudinal imaging of individual tumors to study tumor response and resistance to combinations of ICB, anti-angiogenic therapy, and cisplatin chemotherapy. We found that anti-angiogenic blockade of VEGFA and angiopoietin-2 (ANGPT2) with the bispecific antibody A2V was superior to single VEGFA inhibition and markedly slowed tumor progression. But, contrary to findings in other tumor models, the addition of a PD1 antibody to A2V did not provide additive benefits and even accelerated growth of a fraction of the tumors. We implicated tumor-associated macrophages (TAMs) and PD-1+regulatory T cells (T-regs) in this growth-promoting response, and identified a pre-clinical strategy involving the differential targeting of two distinct TAM subsets that unleashed the therapeutic potential of anti-angiogenic immunotherapy and achieved regression of more than 80% of the lung tumors. Citation Format: Amaia Martinez-Usatorre, Ece Kadioglu, Chiara Cianciaruso, Alan Guichard, Bruno Torchia, Sina Nassiri, Martina Schmittnaegel, Carola H. Ries, Etienne Meylan, Ioanna Keklikoglou, Michele De Palma. Determinants of tumor resistance to anti-angiogenic immunotherapy in mouse models of Kras-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 75.

We describe a lentivirus-encoded chimeric receptor, termed extracellular vesicle (EV)-internalizing receptor (EVIR), which enables the selective uptake of cancer-cell-derived EVs by dendritic cells (DCs). The EVIR enhances DC presentation of EV-associated tumor antigens to CD8+ T cells primarily through MHCI recycling and cross-dressing. EVIRs should facilitate exploring the mechanisms and implications of horizontal transfer of tumor antigens to antigen-presenting cells.

A robust and reproducible method for culturing monolayers of adherent and well-spread primary islet cells on glass coverslips is required for detailed imaging studies by super-resolution and live-cell microscopy. Guided by an observation that dispersed islet cells spread and adhere well on glass surfaces in neuronal co-culture and form a monolayer of connected cells, we demonstrate that in the absence of neurons, well-defined surface coatings combined with components of neuronal culture media collectively support robust attachment and growth of primary human or rat islet cells as monolayers on glass surfaces. The islet cell monolayer cultures on glass stably maintain distinct mono-hormonal insulin+, glucagon+, somatostatin+ and PP+ cells and glucose-responsive synchronized calcium signaling as well as expression of the transcription factors Pdx-1 and NKX-6.1 in beta cells. This technical advance enabled detailed observation of sub-cellular processes in primary human and rat beta cells by super-resolution microscopy. The protocol is envisaged to have broad applicability to sophisticated analyses of pancreatic islet cells that reveal new biological insights, as demonstrated by the identification of an in vitro protocol that markedly increases proliferation of primary beta cells and is associated with a reduction in ciliated, ostensibly proliferation-suppressed beta cells.

A robust and reproducible method for culturing monolayers of adherent and well-spread primary islet cells on glass coverslips is required for detailed imaging studies by super-resolution and live-cell microscopy. Guided by an observation that dispersed islet cells spread and adhere well on glass surfaces in neuronal co-culture and form a monolayer of connected cells, we demonstrate that in the absence of neurons, well-defined surface coatings combined with components of neuronal culture media collectively support robust attachment and growth of primary human or rat islet cells as monolayers on glass surfaces. The islet cell monolayer cultures on glass stably maintain distinct mono-hormonal insulin+, glucagon+, somatostatin+ and PP+ cells and glucose-responsive synchronized calcium signaling as well as expression of the transcription factors Pdx-1 and NKX-6.1 in beta cells. This technical advance enabled detailed observation of sub-cellular processes in primary human and rat beta cells by super-resolution microscopy. The protocol is envisaged to have broad applicability to sophisticated analyses of pancreatic islet cells that reveal new biological insights, as demonstrated by the identification of an in vitro protocol that markedly increases proliferation of primary beta cells and is associated with a reduction in ciliated, ostensibly proliferation-suppressed beta cells.

The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1D), are intracellularmembrane proteins, whose initial encounter with the immune system is poorly understood. Here we propose a new model for how these proteins can initiate autoimmunity. We found that rat and human pancreatic islets release the intracellular β-cell autoantigens in human T1D, GAD65, IA-2, and proinsulin in exosomes, which are taken up by and activate dendritic cells. Accordingly, the anchoring of GAD65 to exosome-mimetic liposomes strongly boosted antigen presentation and T-cell activation in the context of the human T1D susceptibility haplotype HLADR4. Cytokine-induced endoplasmic reticulum stress enhanced exosome secretion by β-cells; induced exosomal release of the immunostimulatory chaperones calreticulin, Gp96, and ORP150; and increased exosomal stimulation of antigen-presenting cells. We propose that stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperonesmay play a role in the initiation of autoimmune responses in T1D. © 2017 by the American Diabetes Association.

The inhibitory neurotransmitter GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD) in neurons and in pancreatic β-cells in islets of Langerhans where it functions as a paracrine and autocrine signaling molecule regulating the function of islet endocrine cells. The localization of the two non-allelic isoforms GAD65 and GAD67 to vesicular membranes is important for rapid delivery and accumulation of GABA for regulated secretion. While the membrane anchoring and trafficking of GAD65 are mediated by intrinsic hydrophobic modifications, GAD67 remains hydrophilic, and yet is targeted to vesicular membrane pathways and synaptic clusters in neurons by both a GAD65-dependent and a distinct GAD65-independent mechanism. Herein we have investigated the membrane association and targeting of GAD67 and GAD65 in monolayer cultures of primary rat, human, and mouse islets and in insulinoma cells. GAD65 is primarily detected in Golgi membranes and in peripheral vesicles distinct from insulin vesicles in β-cells. In the absence of GAD65, GAD67 is in contrast primarily cytosolic in β-cells; its co-expression with GAD65 is necessary for targeting to Golgi membranes and vesicular compartments. Thus, the GAD65-independent mechanism for targeting of GAD67 to synaptic vesicles in neurons is not functional in islet β-cells. Therefore, only GAD65:GAD65 homodimers and GAD67:GAD65 heterodimers, but not the GAD67:GAD67 homodimer gain access to vesicular compartments in β-cells to facilitate rapid accumulation of newly synthesized GABA for regulated secretion and fine tuning of GABA-signaling in islets of Langerhans.

Aim: To study the specificity of cellular MRI based on superparamagnetic iron oxide particles (SPIOs), especially within the CNS. Materials & methods: A microglial cell line was engineered for the expression of a suicide gene, the receptor of diphtheria toxin (DT), and two reporter genes, green fluorescent protein and luciferase, in order to induce, in a controlled manner, cell death and test it through bioluminescence. SPIO-labeled DT-sensitive and control DT-insensitive cells were transplanted into the brains of mice, which underwent serial MRI and bioluminescence studies before and up to 90 days after DT-induced cell death. Results: No variations in SPIO signal voids were detected along longitudinal monitoring in brain hemispheres transplanted with DT-sensitive cells. Ex vivo analyses showed persistence of iron nanoparticle deposits at transplantation sites. Conclusion: Due to the long-term persistence of signal after transplanted cell death, caution is advised when SPIOs are employed for cell tracking. Original submitted 27 January 2014; Revised submitted 18 April 2014

The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1D), are intracellular membrane proteins, whose initial encounter with the immune system is poorly understood. Here we propose a new model for how these proteins can initiate autoimmunity. We found that rat and human pancreatic islets release the intracellular β-cell autoantigens in human T1D, GAD65, IA-2 and proinsulin, in exosomes, which are taken up by and activate dendritic cells. Accordingly, anchoring of GAD65 to exosome-mimetic liposomes strongly boosted antigen presentation and T cell activation in the context of the human type 1 diabetes susceptibility haplotype HLA-DR4. Cytokine-induced ER-stress enhanced exosome secretion by β-cells, induced exosomal release of the immunostimulatory chaperones calreticulin, Gp96 and ORP150 and increased exosomal stimulation of antigen presenting cells. We propose that stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in initiation of autoimmune responses in T1D.