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The interplay among different cells in a tissue is essential for maintaining homeostasis. Although, disease states have been traditionally attributed to individual cell types, increasing evidence and new therapeutic options have demonstrated the primary role of multicellular functions to understand health and disease, opening new avenues to understand pathogenesis and develop new treatment strategies. We recently described the cellular composition and dynamics of the human oral mucosa; however, the spatial arrangement of cells is needed to better understand a morphologically complex tissue. Here, we link single-cell RNA sequencing, spatial transcriptomics, and high-resolution multiplex fluorescence in situ hybridisation to characterise human oral mucosa in health and oral chronic inflammatory disease. We deconvolved expression for resolution enhancement of spatial transcriptomic data and defined highly specialised epithelial and stromal compartments describing location-specific immune programs. Furthermore, we spatially mapped a rare pathogenic fibroblast population localised in a highly immunogenic region, responsible for lymphocyte recruitment through CXCL8 and CXCL10 and with a possible role in pathological angiogenesis through ALOX5AP. Collectively, our study provides a comprehensive reference for the study of oral chronic disease pathogenesis.

The progress made in directed differentiation of stem cells has shown that understanding human pancreas development can provide cues for generating unlimited amounts of insulin-producing beta cells for transplantation therapy in diabetes. However, current differentiation protocols have not been successful in reproducibly generating functional human beta cells in vitro, partly due to incomplete understanding of human pancreas development. Here, we present detailed transcriptomic analysis of the various cell types of the developing human pancreas, including their spatial gene patterns. We integrated single cell RNA sequencing with spatial transcriptomics at multiple developmental timepoints and revealed distinct temporal-spatial gene cascades in the developing human pancreas. Cell trajectory inference identified endocrine progenitor populations and novel branch-specific genes as the progenitors differentiate towards alpha or beta cells, indicating that transcriptional maturation occurred over this developmental timeframe. Spatial differentiation trajectories indicated that immature Schwann cells are spatially co-located with endocrine progenitors and contribute to beta cell maturation via the L1CAM-EPHB2 pathway. Our integrated approach enabled us to identify heterogeneity and multiple lineage dynamics within the mesenchyme, showing that it contributed to the exocrine acinar cell state. Finally, we have generated an interactive web resource for interrogating human pancreas development for the research community.

Current differentiation protocols have not been successful in reproducibly generating fully functional human beta cells in vitro, partly due to incomplete understanding of human pancreas development. Here, we present detailed transcriptomic analysis of the various cell types of the developing human pancreas, including their spatial gene patterns. We integrated single-cell RNA sequencing with spatial transcriptomics at multiple developmental time points and revealed distinct temporal-spatial gene cascades. Cell trajectory inference identified endocrine progenitor populations and branch-specific genes as the progenitors differentiate toward alpha or beta cells. Spatial differentiation trajectories indicated that Schwann cells are spatially co-located with endocrine progenitors, and cell-cell connectivity analysis predicted that they may interact via L1CAM-EPHB2 signaling. Our integrated approach enabled us to identify heterogeneity and multiple lineage dynamics within the mesenchyme, showing that it contributed to the exocrine acinar cell state. Finally, we have generated an interactive web resource for investigating human pancreas development for the research community.

Background Cryptococcus is the most common cause of meningitis in human immunodeficiency virus (HIV)-infected Africans. Despite universal exposure, only 5%–10% of patients with HIV/acquired immune deficiency syndrome and profound CD4+ T-cell depletion develop disseminated cryptococcosis: host genetic factors may play a role. Prior targeted immunogenetic studies in cryptococcosis have comprised few Africans. Methods We analyzed genome-wide single-nucleotide polymorphism (SNP) genotype data from 524 patients of African descent: 243 cases (advanced HIV with cryptococcal antigenemia and/or cryptococcal meningitis) and 281 controls (advanced HIV, no history of cryptococcosis, negative serum cryptococcal antigen). Results Six loci upstream of the colony-stimulating factor 1 (CSF1) gene, encoding macrophage colony-stimulating factor (M-CSF) were associated with susceptibility to cryptococcosis at P, In this first GWAS conducted in cryptococcosis, we describe, replicate and functionally validate novel loci upstream of the colony stimulating factor 1 (CSF1) gene encoding macrophage colony-stimulating factor (M-CSF) as associated with susceptibility to cryptococcosis in HIV-infected South Africans.

The tumour microenvironment plays a crucial role in the growth and progression of cancer and the presence of tumour-associated macrophages (TAMs) is associated with poor prognosis. Recent studies show that TAMs show transcriptomic, phenotypic, functional and geographical diversity. Here we show that a sialylated tumour-associated glycoform of the mucin MUC1, MUC1-ST, through the engagement of Siglec-9 can specifically and independently induce the differentiation of monocytes into TAMs with a unique phenotype. These TAMs can recruit and maintain neutrophils, inhibit the function of T cells, degrade basement membrane allowing for invasion, are inefficient at phagocytosis, and can induce plasma clotting. This novel macrophage phenotype is enriched in the stroma at the edge of breast cancer nests and their presence is associated with poor prognosis in breast cancer patients.

The tumour microenvironment plays a crucial role in the growth and progression of cancer, and the presence of tumour-associated macrophages (TAMs) is associated with poor prognosis. Recent studies have demonstrated that TAMs display transcriptomic, phenotypic, functional and geographical diversity. Here we show that a sialylated tumour-associated glycoform of the mucin MUC1, MUC1-ST, through the engagement of Siglec-9 can specifically and independently induce the differentiation of monocytes into TAMs with a unique phenotype that to the best of our knowledge has not previously been described. These TAMs can recruit and prolong the lifespan of neutrophils, inhibit the function of T cells, degrade basement membrane allowing for invasion, are inefficient at phagocytosis, and can induce plasma clotting. This macrophage phenotype is enriched in the stroma at the edge of breast cancer nests and their presence is associated with poor prognosis in breast cancer patients., Beatson et al. show that a sialylated tumour-associated glycoform of the mucin MUC1 induces the differentiation of monocytes into tumour-associated macrophages. These macrophages are found in breast cancer stroma and their presence is associated with poor prognosis.

Tumor cells engage in bidirectional interactions with stroma and immune cells to promote disease progression and immune evasion. Stroma-specific gene signatures have been associated with outcome in diffuse large B-cell lymphoma (DLBCL), but their immunobiology has been understudied. To characterize the stromal landscape in lymphoma, we performed high-dimensional imaging mass cytometry analysis of the major stroma subsets and revealed a marked expansion and remodeling of the immuno-specialized fibroblastic reticular cells (FRCs) in human DLBCL biopsies (n=53). The FRC network was similarly remodeled in tumors from the IμBcl6 transgenic model of lymphoma, and aberrant fibroblasts were in close proximity to cancer cells. Modelling the interactions between murine and patient FRCs and tumor cells, using 2D and 3D cultures, showed that lymphoma drives the acquisition of an inflammatory-like, pro-tumoral (upregulation of fibroblast activating protein-α, FAP) phenotype and associated functional capabilities. Comparative bulk transcriptomic analysis revealed that lymphoma-FRCs undergo transcriptional reprogramming and activate gene pathways associated with inflammatory responses. Moreover, single-cell RNA-seq revealed an expansion of activated FRC clusters expressing B cell supporting genes, while T cell-associated FRCs were contracted. Altered chemokine signaling pathways in DLBCL-FRCs were functionally linked to reduced attraction of T cells and impeded migration along the lymphoma-reticular network. Moreover, lymphoma-FRCs upregulated expression of inhibitory PD-1 ligands that reduced the anti-tumor cytolytic activity of CD8+ T cells, a T cell bispecific antibody (CD20-TCB, glofitamab) and anti-CD19 CAR T cells in our coculture models. To overcome the immunosuppressive activity of DLBCL-FRCs, we investigated the use of CD20-TCB in combination with stroma-targeting immunocytokine fusion protein drug (FAP-IL2v, RG7461) or costimulatory fusion protein (FAP-4-1BBL, RG7827). Functional cytotoxicity assays using human and murine primary DLBCL patient samples revealed that both stroma-targeting drugs paired effectively with the CD20-TCB to enhance the cytotoxic activity of autologous CD8+ T cells. In addition, the ability of immune-/stroma- targeted combination immunotherapy to trigger anti-tumor activity and CD8+ T cell retention within the FRC-TME was demonstrated using 3D precision-cut lymph node slice-based organotypic cultures of DLBCL and other B cell malignancies. In conclusion our data reveal that lymphoma cells actively reprogram FRCs that acquire altered immunoregulatory function which prevents effective T cell motility and suppresses the anti-tumor function of cytolytic T cells. Importantly, we demonstrate that combination immunotherapy incorporating fibroblast-targeting fusion proteins could effectively recover anti-tumor T cell activity. Citation Format: Benedetta Apollonio, Nedyalko Petrov, Filomena Spada, Peter Jarvis, Domenico Cozzetto, Shichina Kannambath, David Kuo, Mansoor Saqui, Rose-Marie Amini, Gunilla Enblad, Graham Charlotte, Reuben Benjamin, Anna Vardi, Elisabeth Phillips, Jon Salisbury, Eric N. Olson, Brian Fox, Patrick Hagner, Anita Gandhi, Ruth F. Jarrett, Sylvia Herter, Marina Bacac, Christina Klaus, Christian Klein, Alexander Deutsch, Alan G. Ramsay. Stroma-immune landscape in lymphoma: new mechanisms of immunosuppression and therapeutic targeting [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 3165.

RATIONALE: Pulmonary aspergillosis is a lethal mould infection in the immunocompromised host. Understanding initial control of infection, and how this is altered in the immunocompromised host, is a key goal for understanding the pathogenesis of pulmonary aspergillosis. OBJECTIVES: To characterise the outcome of human macrophage infection with Aspergillus fumigatus, and how this is altered in transplant recipients on calcineurin inhibitor immunosuppressants. METHODS: We defined the outcome of human macrophage infection with Aspergillus fumigatus, and the impact of calcineurin inhibitors, through a combination of single cell fluorescence imaging, transcriptomics, proteomics, and in vivo studies. MEASUREMENTS AND MAIN RESULTS: Macrophage phagocytosis of Aspergillus fumigatus enabled control of 90% of fungal germination. However fungal germination in the late phagosome led to macrophage necrosis. During programmed necroptosis, we observed frequent cell-cell transfer of Aspergillus fumigatus between macrophages which assists subsequent control of germination in recipient macrophages. Lateral transfer occurred through actin-dependent exocytosis of the late endosome in a vasodilator-stimulated phosphoprotein (VASP) envelope. Its relevance to the control of fungal germination was also shown by direct visualisation in our zebrafish aspergillosis model in vivo. The calcineurin inhibitor FK506/tacrolimus reduced cell death and lateral transfer in vitro by 50%. This resulted in uncontrolled fungal germination in macrophages and hyphal escape. CONCLUSIONS: These observations identify programmed necrosis-dependent lateral transfer of Aspergillus fumigatus between macrophages as an important host strategy for controlling fungal germination. This process is critically dependent on calcineurin. Our studies provide fundamental insights into the pathogenesis of pulmonary aspergillosis in the immunocompromised host.

Recurrence of meningitis due toCryptococcus neoformansafter treatment causes substantial mortality in HIV/AIDS patients across sub-Saharan Africa. In order to determine whether recurrence occurred due to relapse of the original infecting isolate or reinfection with a different isolate weeks or months after initial treatment, we used whole-genome sequencing to assess the genetic basis of infection in 17 HIV-infected individuals with recurrent cryptococcal meningitis. Comparisons revealed a clonal relationship for 15 pairs of isolates recovered before and after recurrence showing relapse of the original infection. The two remaining pairs showed high levels of genetic heterogeneity; in one pair we found this to be a result of infection by mixed genotypes, whilst the second was a result of nonsense mutations in the gene encoding the DNA mismatch repair proteinsMSH2, MSH5andRAD5. These nonsense mutations led to a hypermutator state, leading to dramatically elevated rates of synonymous and non-synonymous substitutions. Hypermutator phenotypes owing to nonsense mutations in these genes have not previously been reported inCryptococcus neoformansand represent a novel pathway for rapid within-host adaptation and evolution of resistance to firstline antifungal drugs.

Dhh1 and Pat1 in yeast are mRNA decapping activators/\ud translational repressors thought to play key\ud roles in the transition of mRNAs from translation to\ud degradation. However, little is known about the\ud physical and functional relationships between these\ud proteins and the translation machinery. We describe\ud a previously unknown type of diauxic shiftdependent\ud modulation of the intracellular locations\ud of Dhh1 and Pat1. Like the formation of P bodies,\ud this phenomenon changes the spatial relationship\ud between components involved in translation and\ud mRNA degradation. We report significant spatial\ud separation of Dhh1 and Pat1 from ribosomes in\ud exponentially growing cells. Moreover, biochemical\ud analyses reveal that these proteins are excluded\ud from polysomal complexes in exponentially growing\ud cells, indicating that they may not be associated\ud with active states of the translation machinery. In\ud contrast, under diauxic growth shift conditions,\ud Dhh1 and Pat1 are found to co-localize with polysomal\ud complexes. This work suggests that Dhh1\ud and Pat1 functions are modulated by a relocalization\ud mechanism that involves eIF4A. Pulldown\ud experiments reveal that the intracellular\ud binding partners of Dhh1 and Pat1 change as cells\ud undergo the diauxic growth shift. This reveals a\ud new dimension to the relationship between translation\ud activity and interactions between mRNA, the\ud translation machinery and decapping activator\ud proteins.

Invasive fungal infections are a major cause of mortality in solid-organ transplantation where steroids and calcineurin inhibitors form the core of immunosuppression. Our group has previously shown in established hydrocortisone-based mouse models of invasive aspergillosis that calcineurin inhibitors increase mortality through effects on the innate immune response. 1 As alveolar macrophages present the primary resident innate immune cell in the airways responsible for fungal clearance, we perform a detailed study of the role of the calcineurin pathway in the human macrophage response to A. fumigatus (AF). We show that the calcineurin-NFAT pathway is highly activated in the human lung transplant alveolar macrophage response to AF with inhibition resulting in impaired fungal clearance. Calcineurin inhibition leads to delayed phagocytosis, reduction in reactive-oxygen species production and an impairment of a novel actin-dependent process of lateral transfer of swollen AF conidia between human macrophages. Further characterisation reveals that transfer of AF occurs during macrophage necroptosis with subsequently around 50% control of germination in the receiving macrophage. To understand the calcineurin-dependent mechanism, next generation RNA sequencing was performed which confirms that calcineurin inhibition impairs the macrophage programmed cell death immune response. Utilising phosphoproteomics we additionally show that calcineurin inhibition impairs dephosphorylation of vasodilator-stimulated phosphoprotein (VASP), an important actin regulatory protein which promotes actin filament formation. High-resolution confocal microscopy confirms that VASP strongly co-localises to AF conidial phagocytosis and facilitates lateral transfer through tunnel-like structures. Lastly, we utilise a zebrafish model of invasive aspergillosis to confirm the in-vivo relevance of AF macrophage lateral transfer. In conclusion our data shows the importance of the calcineurin pathway in the macrophage innate immune response to AF and highlights a novel calcineurin-actin dependent host defense mechanism which may have significant implications on persistence and dissemination within solid organ transplantation. To our knowledge this is the first report of a host-mediated cell-cell transfer mechanism for any pathogen. Reference 1 Herbst S, Shah A, Mazon Moya M, Marzola V, Jensen B, Reed A, et al . Phagocytosis-dependent activation of a TLR9-BTK-calcineurin-NFAT pathway co-ordinates innate immunity to Aspergillus fumigatus. EMBO Mol Med . 2015; 7 (3):240–58

I-superfamily conotoxins have four-disulfide bonds with cysteine arrangement C-C-CC-CC-C-C, and they inhibit or modify ion channels of nerve cells. They have been characterized only recently and are relatively less well studied compared to other superfamily conotoxins. We have detected selective and sensitive sequence pattern for I-superfamily conotoxins. The availability of sequence pattern should be useful in protein family classification and functional annotation. We have built by homology modeling, a theoretical structural 3D model of ViTx from Conus virgo, a typical member of I-superfamily conotoxins. The modeling was based on the available 3D structure of Janus-atracotoxin-Hv1c of Janus-atracotoxin family whose members have been suggested as possible biopesticides. A study comparing the theoretically modeled structure of ViTx, with experimentally determined structures of other toxins, which share functional similarity with ViTx, reveals the crucial role of C-terminal region of ViTx in blocking therapeutically important voltage-gated potassium channels.