Localized immune surveillance of primary melanoma in the skin deciphered through executable modeling

While skin is a site of active immune surveillance, primary melanomas often escape detection. Here, we have developed an in silico model to determine the local cross-talk between melanomas and Langerhans cells (LCs), the primary antigen-presenting cells at the site of melanoma development. The model predicts that melanomas fail to activate LC migration to lymph nodes until tumors reach a critical size, which is determined by a positive TNF-α feedback loop within melanomas, in line with our observations of murine tumors. In silico drug screening, supported by subsequent experimental testing, shows that treatment of primary tumors with MAPK pathway inhibitors may further prevent LC migration. In addition, our in silico model predicts treatment combinations that bypass LC dysfunction. In conclusion, our combined approach of in silico and in vivo studies suggests a molecular mechanism that explains how early melanomas develop under the radar of immune surveillance by LC

Phenotypic and Functional Characterisation of Hofbauer Cells Across Gestation

The placenta is the first organ the fetus makes and is the interface between the mother and baby. A normal functioning placenta is crucial for a successful pregnancy. As the placenta develops, highly branched villous tree-like structures form which contain fibroblasts, immature capillaries and macrophages, termed Hofbauer cells (HBC). HBC are the only fetal immune cell population found in the healthy placenta throughout pregnancy. The roles of HBC at different stages of pregnancy are unclear, but they are likely to be important in placental development. We sought to provide an in-depth, high-dimensional characterisation of HBC within first trimester and full-term placenta, understand how changes in their function aid placental physiology, and investigate potential factors driving HBC characteristics. Whole-mount immunofluorescence imaging revealed that HBC morphology within placental villi changes during gestation; appearing as rounded cells in the first trimester compared to highly elongated and branched cells at full term. Live ex vivo imaging demonstrated that HBC were motile in the first-trimester placenta and sessile by full term. A high-dimensional mass cytometry (CyTOF) panel was developed and used to show that within the first trimester placenta HBC were a homogeneous population, lacking expression of the HLA class II marker HLA-DR. This contrasted with full-term placenta where HBC displayed heterogeneous HLA- DR expression. Analysis of publicly available 10X Genomics single-cell RNA-sequencing data determined that full-term HBC heterogeneity was restricted to variable HLA-DR expression. Furthermore, a robust bulk RNA-sequencing dataset was generated to profile placental macrophages from the first trimester and full-term placenta. This allowed for in-depth transcriptomic analyses which determined exceptional similarity between full-term HBC subsets. Functional enrichment analyses revealed that first trimester HBC were associated with placental processes such as hormone synthesis, trophoblast migration and vasculogenesis. Contrastingly, full-term HBC were associated with complement regulation, angiogenesis, and branching morphogenesis. An optimised in vitro culture system was established to show that the HLA-DR expression profile of HBC remains unchanged upon culture and HBC are resistant to IFNγ-inducible HLA class II expression. ATAC-seq revealed that chromatin accessibility around HLA class II genes and the master regulator transcription factor CIITA was reduced in first trimester HBC compared to full-term HBC and monocytes. Lastly, monocyte-derived macrophage cultures demonstrated the importance of M-CSF for driving key features of the HBC phenotype. In summary, the results presented in this thesis provide an in-depth characterisation of HBC and how they change across gestation to aid placental function in a healthy pregnancy

The Ontogeny and Function of Placental Macrophages.

The placenta is a fetal-derived organ whose function is crucial for both maternal and fetal health. The human placenta contains a population of fetal macrophages termed Hofbauer cells. These macrophages play diverse roles, aiding in placental development, function and defence. The outer layer of the human placenta is formed by syncytiotrophoblast cells, that fuse to form the syncytium. Adhered to the syncytium at sites of damage, on the maternal side of the placenta, is a population of macrophages termed placenta associated maternal macrophages (PAMM1a). Here we discuss recent developments that have led to renewed insight into our understanding of the ontogeny, phenotype and function of placental macrophages. Finally, we discuss how the application of new technologies within placental research are helping us to further understand these cells

Research data supporting: "Human placental cells are resistant to SARS-CoV-2 infection and replication"

Aim: Quantify the permissiveness of placental cells to SARS-CoV-2 infection and ability to support viral release Data Collection: For tissue processing, decidua was removed and the tissue was washed in PBS for 10min. Villi were scraped and digested in 0.2% Trypsin/0.02% EDTA at 37°C for 7/15min. The digest was filtered through a gauze and quenched with FBS. Undigested material was further digested with 1mg/ml Collagenase V supplemented with 10mg/ml DNase for 30/40min at 37°C. After filtering and washing, layer the cell suspension on to a Pancoll gradient ad centrifuge without break for 20min at 3000rpm. For more details and gating strategies, refer to Appios et al., 2021 (Bio Protoc) and Thomas et al., 2021 (JEM. For tissue sections, slides were fixed and permeabilised in ice-cold acetone (5min), washed in PBS, blocked in 2% serum (20min), incubated overnight at 4°C with primary antibodies - ACE2 (rabbit, ab15348), cytokeratin 7 (mouse, M7018). Slides were washed twice in PBS, and incubated with secondary/conjugated antibodies (anti-rabbit AF488, (A-11008), anti-mouse AF555 (A-31570) and CD31-AF700 (303133)) for 1hr in the dark. Slides were washed twice before staining with DAPI (D9542) and mounted ready for imaging. For in vitro assays, cells were infected with BetaCoV (B.1.1.7) strain at MOI 1 for 1hr. Inoculum was removed and cells washed with PBS before replacing with media containing 2% serum. Cells were fixed at 24h in 4% PFA. Cells were permeabilised (0.1% saponin, 10min), and blocked (1% BSA and 0.01% saponin, 20min) before incubating with the primary antibody SARS-CoV-2 spike protein (mouse, GTX632604) for 1hr. After 3 washes (5min each) in blocking solution, cells were incubated with the secondary antibody, anti-mouse AF488 (A-32766) for 45min in the dark. After washing 3 times, nuclei were stained with Hoechst 33342 (62249) for 5min, washed with PBS and maintained in PBS ready for imaging. Data generation: Human primary Hofbauer cells were sorted and data recorded on the BD FACS Aria III using the BD FACSDiva™ software. Tissue sections and cells from infection assays were imaged using the Zeiss LSM780 AxioObserver. Brightness and contrast of images were adjusted using the Brightness/Contrast tool in the FiJi (Just ImageJ) software

Additional Research data supporting "Human placental cells are resistant to SARS-CoV-2 infection and replication"

Aim: Quantify the permissiveness of placental cells to SARS-CoV-2 infection and ability to support viral release Data Collection: For tissue processing, decidua was removed and the tissue was washed in PBS for 10min. Villi were scraped and digested in 0.2% Trypsin/0.02% EDTA at 37°C for 7/15min. The digest was filtered through a gauze and quenched with FBS. Undigested material was further digested with 1mg/ml Collagenase V supplemented with 10mg/ml DNase for 30/40min at 37°C. After filtering and washing, layer the cell suspension on to a Pancoll gradient ad centrifuge without break for 20min at 3000rpm. For more details and gating strategies, refer to Appios et al., 2021 (Bio Protoc) and Thomas et al., 2021 (JEM. For tissue sections, slides were fixed and permeabilised in ice-cold acetone (5min), washed in PBS, blocked in 2% serum (20min), incubated overnight at 4°C with primary antibodies - ACE2 (rabbit, ab15348), cytokeratin 7 (mouse, M7018). Slides were washed twice in PBS, and incubated with secondary/conjugated antibodies (anti-rabbit AF488, (A-11008), anti-mouse AF555 (A-31570) and CD31-AF700 (303133)) for 1hr in the dark. Slides were washed twice before staining with DAPI (D9542) and mounted ready for imaging. For in vitro assays, cells were infected with BetaCoV (B.1.1.7) strain at MOI 1 for 1hr. Inoculum was removed and cells washed with PBS before replacing with media containing 2% serum. Cells were fixed at 24h in 4% PFA. Cells were permeabilised (0.1% saponin, 10min), and blocked (1% BSA and 0.01% saponin, 20min) before incubating with the primary antibody SARS-CoV-2 spike protein (mouse, GTX632604) for 1hr. After 3 washes (5min each) in blocking solution, cells were incubated with the secondary antibody, anti-mouse AF488 (A-32766) for 45min in the dark. After washing 3 times, nuclei were stained with Hoechst 33342 (62249) for 5min, washed with PBS and maintained in PBS ready for imaging. Data generation: Human primary Hofbauer cells were sorted and data recorded on the BD FACS Aria III using the BD FACSDiva™ software. Tissue sections and cells from infection assays were imaged using the Zeiss LSM780 AxioObserver. Brightness and contrast of images were adjusted using the Brightness/Contrast tool in the FiJi (Just ImageJ) software. This data is linked to the repository (https://doi.org/10.17863/CAM.106485) and provides the additional raw .lsm files of all images used in the paper (https://doi.org/10.12688/wellcomeopenres.20514.1).Wellcome Trust (204464/Z/16/A

Primitive haematopoiesis in the human placenta gives rise to macrophages with epigenetically silenced HLA-DR.

The earliest macrophages are generated during embryonic development from erythro-myeloid progenitors (EMPs) via primitive haematopoiesis. Although this process is thought to be spatially restricted to the yolk sac in the mouse, in humans, it remains poorly understood. Human foetal placental macrophages, or Hofbauer cells (HBC), arise during the primitive haematopoietic wave ~18 days post conception and lack expression of human leukocyte antigen (HLA) class II. Here, we identify a population of placental erythro-myeloid progenitors (PEMPs) in the early human placenta that have conserved features of primitive yolk sac EMPs, including the lack of HLF expression. Using in vitro culture experiments we demonstrate that PEMP generate HBC-like cells lacking HLA-DR expression. We find the absence of HLA-DR in primitive macrophages is mediated via epigenetic silencing of class II transactivator, CIITA, the master regulator of HLA class II gene expression. These findings establish the human placenta as an additional site of primitive haematopoiesis

A Crohn’s Disease-associated IL2RA Enhancer Variant Determines the Balance of T Cell Immunity by Regulating Responsiveness to IL-2 Signalling

BACKGROUND AND AIMS: Differential responsiveness to interleukin [IL]-2 between effector CD4(+) T cells [T(eff)] and regulatory T cells [T(reg)] is a fundamental mechanism of immunoregulation. The single nucleotide polymorphism [SNP] rs61839660, located within IL2RA [CD25], has been associated with the development of Crohn’s disease [CD]. We sought to identify the T cell immune phenotype of IBD patients who carry this SNP. METHODS: T(eff) and T(reg) were isolated from individuals homozygous [TT], heterozygous [CT], or wild-type [CC] for the minor allele at rs61839660, and used for phenotyping [flow cytometry, Cytometry Time Of Flight] functional assays or T cell receptor [TCR] sequencing. Phosphorylation of signal transducer and activator of transcription 5 [STAT5] was assessed in response to IL-2, IL-7, and in the presence of basiliximab, a monoclonal antibody directed against CD25. T(eff) pro-inflammatory cytokine expression levels were assessed by reverse transcription quantitative polymerase chain reaction after IL-2 and/or TCR stimulation. RESULTS: Presence of the minor T allele enhances CD25 expression, leading to increased STAT5 phosphorylation and pro-inflammatory cytokine transcript expression by T(eff) in response to IL-2 stimulation in vitro. T(eff) from TT individuals demonstrate a more activated gut homing phenotype. TCR sequencing analysis suggests that TT patients may have a reduced clonal capacity to mount an optimal regulatory T cell response. CONCLUSIONS: rs61839660 regulates the responsiveness of T cells to IL-2 signalling by modulating CD25 expression. As low-dose IL-2 is being trialled as a selective T(reg) modulator in CD, these findings highlight the potential for adverse effects in patients with this genotype

Phenotypic and functional characterization of first-trimester human placental macrophages, Hofbauer cells.

Hofbauer cells (HBCs) are a population of macrophages found in high abundance within the stroma of the first-trimester human placenta. HBCs are the only fetal immune cell population within the stroma of healthy placenta. However, the functional properties of these cells are poorly described. Aligning with their predicted origin via primitive hematopoiesis, we find that HBCs are transcriptionally similar to yolk sac macrophages. Phenotypically, HBCs can be identified as HLA-DR-FOLR2+ macrophages. We identify a number of factors that HBCs secrete (including OPN and MMP-9) that could affect placental angiogenesis and remodeling. We determine that HBCs have the capacity to play a defensive role, where they are responsive to Toll-like receptor stimulation and are microbicidal. Finally, we also identify a population of placenta-associated maternal macrophages (PAMM1a) that adhere to the placental surface and express factors, such as fibronectin, that may aid in repair.WT grant number 204464/Z/16/Z, WT grant number 215226/Z/19/Z, MRC grant number: MR/P001092/