Recent Advances in Our Understanding of Human Inflammatory Dendritic Cells in Human Immunodeficiency Virus Infection.

Anogenital inflammation is a critical risk factor for HIV acquisition. The primary preventative HIV intervention, pre-exposure prophylaxis (PrEP), is ineffective in blocking transmission in anogenital inflammation. Pre-existing sexually transmitted diseases (STIs) and anogenital microbiota dysbiosis are the leading causes of inflammation, where inflammation is extensive and often asymptomatic and undiagnosed.

The spatial biology of HIV infection.

HIV infection implicates a spectrum of tissues in the human body starting with viral transmission in the anogenital tract and subsequently persisting in lymphoid tissues and brain. Though studies using isolated cells have contributed significantly towards our understanding of HIV infection, the tissue microenvironment is characterised by a complex interplay of a range of factors, all of which can influence the course of infection but are otherwise missed in ex vivo studies. To address this knowledge gap, it is necessary to investigate the dynamics of infection and the host immune response in situ using imaging-based approaches.

Characterising plasmacytoid and myeloid AXL+ SIGLEC-6+ dendritic cell functions and their interactions with HIV.

AXL+ Siglec-6+ dendritic cells (ASDC) are novel myeloid DCs which can be subdivided into CD11c+ and CD123+ expressing subsets. We showed for the first time that these two ASDC subsets are present in inflamed human anogenital tissues where HIV transmission occurs. Their presence in inflamed tissues was supported by single cell RNA analysis of public databases of such tissues including psoriasis diseased skin and colorectal cancer.

Herpes simplex virus spreads rapidly in human foreskin, partly driven by chemokine-induced redistribution of Nectin-1 on keratinocytes.

HSV infects keratinocytes in the epidermis of skin via nectin-1. We established a human foreskin explant infection model to investigate HSV entry and spread. HSV1 entry could only be achieved by the topical application of virus via high density microarray projections (HD-MAPs) to the epidermis, which penetrated beyond one third of its thickness, simulating in vivo microtrauma.

OMIP-103: A 35-marker imaging mass cytometry panel for the co-detection of HIV and immune cell populations in human formalin fixed paraffin embedded intestinal tissue.

We introduce a 35-marker imaging mass cytometry (IMC) panel for a detailed examination of immune cell populations and HIV RNA in formalin fixed paraffin embedded (FFPE) human intestinal tissue. The panel has broad cell type coverage and particularly excels in delineating subsets of mononuclear phagocytes and T cells. Markers for key tissue structures are included, enabling identification of epithelium, blood vessels, lymphatics, and musculature.

Imaging Mass Cytometry for In Situ Immune Profiling.

The complexities and cellular heterogeneity associated with tissues necessitate the concurrent detection of markers beyond the limitations of conventional imaging approaches in order to spatially resolve the relationships between immune cell populations and their environments. This is a necessary complement to single-cell suspension-based methods to inform a better understanding of the events that may underlie pathological conditions. Imaging mass cytometry is a high-dimensional imaging modality that allows for the concurrent detection of up to 40 protein markers of interest across tissues at subcellular resolution.

Epithelial dendritic cells vs. Langerhans cells: Implications for mucosal vaccines.

Next-generation vaccines may be delivered via the skin and mucosa. The stratified squamous epithelium (SSE) represents the outermost layer of the skin (epidermis) and type II mucosa (epithelium). Langerhans cells (LCs) have been considered the sole antigen-presenting cells (APCs) to inhabit the SSE; however, it is now clear that dendritic cells (DCs) are also present.

Nerve-myeloid cell interactions in persistent human pain: a reappraisal using updated cell subset classifications.

The past 20 years have seen a dramatic shift in our understanding of the role of the immune system in initiating and maintaining pain. Myeloid cells, including macrophages, dendritic cells, Langerhans cells, and mast cells, are increasingly implicated in bidirectional interactions with nerve fibres in rodent pain models. However, our understanding of the human setting is still poor.

OMIP-096: A 24-color flow cytometry panel to identify and characterize CD4+ and CD8+ tissue-resident T cells in human skin, intestinal, and type II mucosal tissue.

There is a great need to understand human immune cells within tissue, where disease manifests and infection occurs. Tissue-resident memory T cells (TRMs) were discovered over a decade ago, there is a great need to understand their role in human disease. We developed a 24-color flow cytometry panel to comprehensively interrogate CD4 and CD8 TRMs isolated from human tissues.

Spatial analysis for highly multiplexed imaging data to identify tissue microenvironments.

Highly multiplexed in situ imaging cytometry assays have made it possible to study the spatial organization of numerous cell types simultaneously. We have addressed the challenge of quantifying complex multi-cellular relationships by proposing a statistical method which clusters local indicators of spatial association. Our approach successfully identifies distinct tissue architectures in datasets generated from three state-of-the-art high-parameter assays demonstrating its value in summarizing the information-rich data generated from these technologies.

An in situ analysis pipeline for initial host-pathogen interactions reveals signatures of human colorectal HIV transmission.

The initial immune response to HIV determines transmission. However, due to technical limitations we still do not have a comparative map of early mucosal transmission events. By combining RNAscope, cyclic immunofluorescence, and image analysis tools, we quantify HIV transmission signatures in intact human colorectal explants within 2 h of topical exposure.

spicyR: spatial analysis of in situ cytometry data in R.

Motivation: High parameter histological techniques have allowed for the identification of a variety of distinct cell types within an image, providing a comprehensive overview of the tissue environment. This allows the complex cellular architecture and environment of diseased tissue to be explored. While spatial analysis techniques have revealed how cell-cell interactions are important within the disease pathology, there remains a gap in exploring changes in these interactions within the disease process.

HIV transmitting mononuclear phagocytes; integrating the old and new.

In tissue, mononuclear phagocytes (MNP) are comprised of Langerhans cells, dendritic cells, macrophages and monocyte-derived cells. They are the first immune cells to encounter HIV during transmission and transmit the virus to CD4 T cells as a consequence of their antigen presenting cell function. To understand the role these cells play in transmission, their phenotypic and functional characterisation is important.

OMIP 082: A 25-color phenotyping to define human innate lymphoid cells, natural killer cells, mucosal-associated invariant T cells, and γδ T cells from freshly isolated human intestinal tissue.

We developed a 25-color flow cytometry panel to comprehensively interrogate innate lymphoid cells (ILC), mucosal-associated invariant T (MAIT) cells, natural killer (NK) cells and γδ T cells in human tissues. The ability to isolate and interrogate these cells from fresh human tissue is crucial in understanding the role these cells play at immune-privileged mucosal surfaces like the intestine in health and disease settings. However, liberating these cells from tissue is extremely challenging as many key surface identification markers are susceptible to enzymatic cleavage.

Transcriptomic Analysis Identifies A Tolerogenic Dendritic Cell Signature.

Dendritic cells (DC) are central to regulating innate and adaptive immune responses. Strategies that modify DC function provide new therapeutic opportunities in autoimmune diseases and transplantation. Current pharmacological approaches can alter DC phenotype to induce tolerogenic DC (tolDC), a maturation-resistant DC subset capable of directing a regulatory immune response that are being explored in current clinical trials.

Neutrophil Extracellular Trap Density Increases With Increasing Histopathological Severity of Crohn's Disease.

Background: Intestinal neutrophil recruitment is a characteristic feature of the earliest stages of inflammatory bowel disease (IBD). Neutrophil elastase (NE) and myeloperoxidase (MPO) mediate the formation of neutrophil extracellular traps (NETs); NETs produce the bactericidal oxidant hypochlorous acid (HOCl), causing host tissue damage when unregulated. The project aim was to investigate the relationship between NET formation and clinical IBD in humans.

Optimal Isolation Protocols for Examining and Interrogating Mononuclear Phagocytes From Human Intestinal Tissue.

The human intestine contains numerous mononuclear phagocytes (MNP), including subsets of conventional dendritic cells (cDC), macrophages (Mf) and monocytes, each playing their own unique role within the intestinal immune system and homeostasis. The ability to isolate and interrogate MNPs from fresh human tissue is crucial if we are to understand the role of these cells in homeostasis, disease settings and immunotherapies. However, liberating these cells from tissue is problematic as many of the key surface identification markers they express are susceptible to enzymatic cleavage and they are highly susceptible to cell death.

Identification of Genes Encoding Antimicrobial Proteins in Langerhans Cells.

Langerhans cells (LCs) reside in the epidermis where they are poised to mount an antimicrobial response against microbial pathogens invading from the outside environment. To elucidate potential pathways by which LCs contribute to host defense, we mined published LC transcriptomes deposited in GEO and the scientific literature for genes that participate in antimicrobial responses. Overall, we identified 31 genes in LCs that encode proteins that contribute to antimicrobial activity, ten of which were cross-validated in at least two separate experiments.

Herpes Simplex Virus type 1 infects Langerhans cells and the novel epidermal dendritic cell, Epi-cDC2s, via different entry pathways.

Skin mononuclear phagocytes (MNPs) provide the first interactions of invading viruses with the immune system. In addition to Langerhans cells (LCs), we recently described a second epidermal MNP population, Epi-cDC2s, in human anogenital epidermis that is closely related to dermal conventional dendritic cells type 2 (cDC2) and can be preferentially infected by HIV. Here we show that in epidermal explants topically infected with herpes simplex virus (HSV-1), both LCs and Epi-cDC2s interact with HSV-1 particles and infected keratinocytes.

Plasmacytoid dendritic cells have divergent effects on HIV infection of initial target cells and induce a pro-retention phenotype.

Although HIV infection inhibits interferon responses in its target cells in vitro, interferon signatures can be detected in vivo soon after sexual transmission, mainly attributed to plasmacytoid dendritic cells (pDCs). In this study, we examined the physiological contributions of pDCs to early HIV acquisition using coculture models of pDCs with myeloid DCs, macrophages and the resting central, transitional and effector memory CD4 T cell subsets. pDCs impacted infection in a cell-specific manner.