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Article ; Online: 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.

O'Neil, Thomas R / Harman, Andrew N / Cunningham, Anthony L / Nasr, Najla / Bertram, Kirstie M

Cytometry. Part A : the journal of the International Society for Analytical Cytology

2023 Volume 103, Issue 11, Page(s) 851–856

Abstract: 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. ... ...

Abstract	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
MeSH term(s)	Female ; Humans ; Flow Cytometry ; CD8-Positive T-Lymphocytes ; Intestines ; CD4-Positive T-Lymphocytes ; Mucous Membrane ; Immunologic Memory
Language	English
Publishing date	2023-09-29
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2099868-5
ISSN	1552-4930 ; 0196-4763 ; 1552-4922
ISSN (online)	1552-4930
ISSN	0196-4763 ; 1552-4922
DOI	10.1002/cyto.a.24782
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Article ; Online: Defining the landscape of human epidermal mononuclear phagocytes.

Bertram, Kirstie M / O'Neil, Thomas R / Vine, Erica E / Baharlou, Heeva / Cunningham, Anthony L / Harman, Andrew N

Immunity

2023 Volume 56, Issue 3, Page(s) 459–460

MeSH term(s)	Humans ; Dendritic Cells ; Epidermis ; Skin ; Langerhans Cells ; Phagocytes
Language	English
Publishing date	2023-04-05
Publishing country	United States
Document type	Letter ; Comment
ZDB-ID	1217235-2
ISSN	1097-4180 ; 1074-7613
ISSN (online)	1097-4180
ISSN	1074-7613
DOI	10.1016/j.immuni.2023.02.001
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Zs.A 4227: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (2.OG) ab Jg. 2022: Lesesaal (EG)
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Article ; Online: HIV transmitting mononuclear phagocytes; integrating the old and new.

Vine, Erica E / Rhodes, Jake W / Warner van Dijk, Freja A / Byrne, Scott N / Bertram, Kirstie M / Cunningham, Anthony L / Harman, Andrew N

Mucosal immunology

2022 Volume 15, Issue 4, Page(s) 542–550

Abstract: 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 ... ...

Abstract	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. With advancements in high parameter single cell technologies, new MNPs subsets are continuously being discovered and their definition and classification is in a state of flux. This has important implications for our knowledge of HIV transmission, which requires a deeper understanding to design effective vaccines and better blocking strategies. Here we review the historical research of the role MNPs play in HIV transmission up to the present day and revaluate these studies in the context of our most recent understandings of the MNP system.
MeSH term(s)	CD4-Positive T-Lymphocytes ; Dendritic Cells ; HIV Infections ; Humans ; Langerhans Cells ; Macrophages ; Mononuclear Phagocyte System ; Phagocytes
Language	English
Publishing date	2022-02-16
Publishing country	United States
Document type	Journal Article ; Review
ZDB-ID	2411370-0
ISSN	1935-3456 ; 1933-0219
ISSN (online)	1935-3456
ISSN	1933-0219
DOI	10.1038/s41385-022-00492-0
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Article ; Online: 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.

Doyle, Chloe M / Fewings, Nicole L / Ctercteko, Grahame / Byrne, Scott N / Harman, Andrew N / Bertram, Kirstie M

Cytometry. Part A : the journal of the International Society for Analytical Cytology

2022 Volume 101, Issue 3, Page(s) 196–202

Abstract: 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 ... ...

Abstract	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. Choosing the correct enzyme-antibody clone combination within a high-parameter panel is, therefore, a critical consideration. Here, we present a comprehensive, in-depth analysis of the effect different common digestive enzyme blends have on key surface markers used to identify these cell types. In addition, we compared multiple antibody clones for surface markers that are highly susceptible to enzymatic cleavage, such as CD127 and NKp44, to achieve the most consistent and superior staining patterns among donors.
MeSH term(s)	Biomarkers ; Flow Cytometry ; Humans ; Immunity, Innate ; Intestines ; Killer Cells, Natural ; Mucosal-Associated Invariant T Cells
Chemical Substances	Biomarkers
Language	English
Publishing date	2022-01-12
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2099868-5
ISSN	1552-4930 ; 0196-4763 ; 1552-4922
ISSN (online)	1552-4930
ISSN	0196-4763 ; 1552-4922
DOI	10.1002/cyto.a.24529
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Zs.A 1688: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (1.OG) ab Jg. 2022: Lesesaal (EG)
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Article ; Online: AFid: a tool for automated identification and exclusion of autofluorescent objects from microscopy images.

Baharlou, Heeva / Canete, Nicolas P / Bertram, Kirstie M / Sandgren, Kerrie J / Cunningham, Anthony L / Harman, Andrew N / Patrick, Ellis

Bioinformatics (Oxford, England)

2020 Volume 37, Issue 4, Page(s) 559–567

Abstract: Motivation: Autofluorescence is a long-standing problem that has hindered the analysis of images of tissues acquired by fluorescence microscopy. Current approaches to mitigate autofluorescence in tissue are lab-based and involve either chemical ... ...

Abstract	Motivation: Autofluorescence is a long-standing problem that has hindered the analysis of images of tissues acquired by fluorescence microscopy. Current approaches to mitigate autofluorescence in tissue are lab-based and involve either chemical treatment of sections or specialized instrumentation and software to 'unmix' autofluorescent signals. Importantly, these approaches are pre-emptive and there are currently no methods to deal with autofluorescence in acquired fluorescence microscopy images. Results: To address this, we developed Autofluorescence Identifier (AFid). AFid identifies autofluorescent pixels as discrete objects in multi-channel images post-acquisition. These objects can then be tagged for exclusion from downstream analysis. We validated AFid using images of FFPE human colorectal tissue stained for common immune markers. Further, we demonstrate its utility for image analysis where its implementation allows the accurate measurement of HIV-Dendritic cell interactions in a colorectal explant model of HIV transmission. Therefore, AFid represents a major leap forward in the extraction of useful data from images plagued by autofluorescence by offering an approach that is easily incorporated into existing workflows and that can be used with various samples, staining panels and image acquisition methods. We have implemented AFid in ImageJ, Matlab and R to accommodate the diverse image analysis community. Availability and implementation: AFid software is available at https://ellispatrick.github.io/AFid. Supplementary information: Supplementary data are available at Bioinformatics online.
MeSH term(s)	Histological Techniques ; Humans ; Image Processing, Computer-Assisted ; Microscopy, Fluorescence ; Software ; Workflow
Language	English
Publishing date	2020-10-24
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1422668-6
ISSN	1367-4811 ; 1367-4803
ISSN (online)	1367-4811
ISSN	1367-4803
DOI	10.1093/bioinformatics/btaa780
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Article ; Online: Herpes Simplex Virus type 1 infects Langerhans cells and the novel epidermal dendritic cell, Epi-cDC2s, via different entry pathways.

Bertram, Kirstie M / Truong, Naomi R / Smith, Jacinta B / Kim, Min / Sandgren, Kerrie J / Feng, Konrad L / Herbert, Jason J / Rana, Hafsa / Danastas, Kevin / Miranda-Saksena, Monica / Rhodes, Jake W / Patrick, Ellis / Cohen, Ralph C / Lim, Jake / Merten, Steven L / Harman, Andrew N / Cunningham, Anthony L

PLoS pathogens

2021 Volume 17, Issue 4, Page(s) e1009536

Abstract: 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 ...

Abstract	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. Isolated Epi-cDC2s support higher levels of infection than LCs in vitro, inhibited by acyclovir, but both MNP subtypes express similar levels of the HSV entry receptors nectin-1 and HVEM, and show similar levels of initial uptake. Using inhibitors of endosomal acidification, actin and cholesterol, we found that HSV-1 utilises different entry pathways in each cell type. HSV-1 predominantly infects LCs, and monocyte-derived MNPs, via a pH-dependent pathway. In contrast, Epi-cDC2s are mainly infected via a pH-independent pathway which may contribute to the enhanced infection of Epi-cDC2s. Both cells underwent apoptosis suggesting that Epi-cDC2s may follow the same dermal migration and uptake by dermal MNPs that we have previously shown for LCs. Thus, we hypothesize that the uptake of HSV and infection of Epi-cDC2s will stimulate immune responses via a different pathway to LCs, which in future may help guide HSV vaccine development and adjuvant targeting.
MeSH term(s)	Adolescent ; Animals ; Cells, Cultured ; Child ; Child, Preschool ; Chlorocebus aethiops ; Epidermis/pathology ; Epidermis/virology ; HaCaT Cells ; HeLa Cells ; Herpes Simplex/pathology ; Herpes Simplex/virology ; Herpesvirus 1, Human/physiology ; Humans ; Infant ; Langerhans Cells/virology ; Signal Transduction/physiology ; Vero Cells ; Virus Internalization
Language	English
Publishing date	2021-04-27
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2205412-1
ISSN	1553-7374 ; 1553-7366
ISSN (online)	1553-7374
ISSN	1553-7366
DOI	10.1371/journal.ppat.1009536
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Article ; Online: Optimal Isolation Protocols for Examining and Interrogating Mononuclear Phagocytes From Human Intestinal Tissue.

Doyle, Chloe M / Vine, Erica E / Bertram, Kirstie M / Baharlou, Heeva / Rhodes, Jake W / Dervish, Suat / Gosselink, Martijn P / Di Re, Angelina / Collins, Geoffrey P / Reza, Faizur / Toh, James W T / Pathma-Nathan, Nimalan / Ahlenstiel, Golo / Ctercteko, Grahame / Cunningham, Anthony L / Harman, Andrew N / Byrne, Scott N

Frontiers in immunology

2021 Volume 12, Page(s) 727952

Abstract: 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 ... ...

Abstract	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. In addition, the extraction process triggers immunological activation/maturation which alters their functional phenotype. Identifying the evolving, complex and highly heterogenous repertoire of MNPs by flow cytometry therefore requires careful selection of digestive enzyme blends that liberate viable cells and preserve recognition epitopes involving careful selection of antibody clones to enable analysis and sorting for functional assays. Here we describe a method for the anatomical separation of mucosa and submucosa as well as isolating lymphoid follicles from human jejunum, ileum and colon. We also describe in detail the optimised enzyme digestion methods needed to acquire functionally immature and biologically functional intestinal MNPs. A comprehensive list of screened antibody clones is also presented which allows for the development of high parameter flow cytometry panels to discriminate all currently identified human tissue MNP subsets including pDCs, cDC1, cDC2 (langerin
MeSH term(s)	Biomarkers/metabolism ; Cell Separation ; Cells, Cultured ; Colon/cytology ; Cytokines/metabolism ; Dendritic Cells/immunology ; Dendritic Cells/metabolism ; Flow Cytometry ; Humans ; Ileum/cytology ; Intestinal Mucosa/cytology ; Jejunum/cytology ; Macrophages/immunology ; Macrophages/metabolism ; Monocytes/immunology ; Monocytes/metabolism ; Phagocytes/immunology ; Phagocytes/metabolism ; Phenotype
Chemical Substances	Biomarkers ; Cytokines
Language	English
Publishing date	2021-09-01
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2606827-8
ISSN	1664-3224 ; 1664-3224
ISSN (online)	1664-3224
ISSN	1664-3224
DOI	10.3389/fimmu.2021.727952
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Article ; Online: An in situ analysis pipeline for initial host-pathogen interactions reveals signatures of human colorectal HIV transmission.

Cell reports

2022 Volume 40, Issue 12, Page(s) 111385

Abstract: 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 ... ...

Abstract	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. We map HIV enrichment to mucosal dendritic cells (DCs) and submucosal macrophages, but not CD4
MeSH term(s)	CD4-Positive T-Lymphocytes ; Colorectal Neoplasms/pathology ; Dendritic Cells ; HIV Infections ; HIV-1 ; Host-Pathogen Interactions ; Humans
Language	English
Publishing date	2022-09-06
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2649101-1
ISSN	2211-1247 ; 2211-1247
ISSN (online)	2211-1247
ISSN	2211-1247
DOI	10.1016/j.celrep.2022.111385
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Article ; Online: Langerhans cells and sexual transmission of HIV and HSV.

Botting, Rachel A / Rana, Hafsa / Bertram, Kirstie M / Rhodes, Jake W / Baharlou, Heeva / Nasr, Najla / Cunningham, Anthony L / Harman, Andrew N

Reviews in medical virology

2017 Volume 27, Issue 2

Abstract: Langerhans cells (LCs) situated in stratified squamous epithelium of the skin and mucosal tissue are amongst the first cells that sexually transmitted pathogens encounter during transmission. They are potent antigen presenting cells and play a key role ... ...

Abstract	Langerhans cells (LCs) situated in stratified squamous epithelium of the skin and mucosal tissue are amongst the first cells that sexually transmitted pathogens encounter during transmission. They are potent antigen presenting cells and play a key role in the host mounting an appropriate immune response. As such, viruses have evolved complex strategies to manipulate these cells to facilitate successful transmission. One of best studied examples is HIV, which manipulates the natural function of these cells to interact with CD4 T cells, which are the main target cell for HIV in which rapid replication occurs. However, there is controversy in the literature as to the role that LCs play in this process. Langerhans cells also play a key role in the way the body mounts an immune response to HSV, and there is also a complex interplay between the transmission of HSV and HIV that involves LCs. In this article, we review both past and present literatures with a particular focus on a few very recent studies that shed new light on the role that LCs play in the transmission and immune response to these 2 pathogens.
MeSH term(s)	HIV Infections/transmission ; Herpes Genitalis/transmission ; Host-Pathogen Interactions ; Humans ; Langerhans Cells/immunology ; Langerhans Cells/virology
Language	English
Publishing date	2017-03
Publishing country	England
Document type	Journal Article ; Review
ZDB-ID	1086043-5
ISSN	1099-1654 ; 1052-9276
ISSN (online)	1099-1654
ISSN	1052-9276
DOI	10.1002/rmv.1923
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Zs.A 3308: Show issues

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Article ; Online: Herpes simplex virus type 2-infected dendritic cells produce TNF-α, which enhances CCR5 expression and stimulates HIV production from adjacent infected cells.

Marsden, Valerie / Donaghy, Heather / Bertram, Kirstie M / Harman, Andrew N / Nasr, Najla / Keoshkerian, Elizabeth / Merten, Steven / Lloyd, Andrew R / Cunningham, Anthony L

Journal of immunology (Baltimore, Md. : 1950)

2015 Volume 194, Issue 9, Page(s) 4438–4445

Abstract: Prior HSV-2 infection enhances the acquisition of HIV-1 >3-fold. In genital herpes lesions, the superficial layers of stratified squamous epithelium are disrupted, allowing easier access of HIV-1 to Langerhans cells (LC) in the epidermis and perhaps even ...

Abstract	Prior HSV-2 infection enhances the acquisition of HIV-1 >3-fold. In genital herpes lesions, the superficial layers of stratified squamous epithelium are disrupted, allowing easier access of HIV-1 to Langerhans cells (LC) in the epidermis and perhaps even dendritic cells (DCs) in the outer dermis, as well as to lesion infiltrating activated T lymphocytes and macrophages. Therefore, we examined the effects of coinfection with HIV-1 and HSV-2 on monocyte-derived DCs (MDDC). With simultaneous coinfection, HSV-2 significantly stimulated HIV-1 DNA production 5-fold compared with HIV-1 infection alone. Because <1% of cells were dually infected, this was a field effect. Virus-stripped supernatants from HSV-2-infected MDDCs were shown to enhance HIV-1 infection, as measured by HIV-1-DNA and p24 Ag in MDDCs. Furthermore these supernatants markedly stimulated CCR5 expression on both MDDCs and LCs. TNF-α was by far the most prominent cytokine in the supernatant and also within HSV-2-infected MDDCs. HSV-2 infection of isolated immature epidermal LCs, but not keratinocytes, also produced TNF-α (and low levels of IFN-β). Neutralizing Ab to TNF-α and its receptor, TNF-R1, on MDDCs markedly inhibited the CCR5-stimulating effect of the supernatant. Therefore, these results suggest that HSV-2 infection of DCs in the skin during primary or recurrent genital herpes may enhance HIV-1 infection of adjacent DCs, thus contributing to acquisition of HIV-1 through herpetic lesions.
MeSH term(s)	Coinfection ; Culture Media, Conditioned/metabolism ; Cytokines/biosynthesis ; Dendritic Cells/immunology ; Dendritic Cells/metabolism ; Dendritic Cells/virology ; Gene Expression Regulation ; HIV Infections/genetics ; HIV Infections/immunology ; HIV Infections/metabolism ; HIV Infections/virology ; HIV-1/physiology ; Herpes Genitalis/genetics ; Herpes Genitalis/immunology ; Herpes Genitalis/metabolism ; Herpesvirus 2, Human/physiology ; Herpesvirus 2, Human/radiation effects ; Humans ; Models, Biological ; Receptors, CCR5/genetics ; Receptors, CCR5/metabolism ; Tumor Necrosis Factor-alpha/metabolism ; Up-Regulation ; Virus Replication
Chemical Substances	Culture Media, Conditioned ; Cytokines ; Receptors, CCR5 ; Tumor Necrosis Factor-alpha
Language	English
Publishing date	2015-05-01
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	3056-9
ISSN	1550-6606 ; 0022-1767 ; 1048-3233 ; 1047-7381
ISSN (online)	1550-6606
ISSN	0022-1767 ; 1048-3233 ; 1047-7381
DOI	10.4049/jimmunol.1401706
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