Seven-colour multiplex immunochemistry/immunofluorescence and whole slide imaging of frozen sections.

Multiplex Immunochemistry/Immunofluorescence (mIHC/IF) aims to visualise multiple biomarkers in a single tissue section and is especially powerful when used on slide scanners coupled with digital analysis tools. mIHC/IF is commonly employed in immuno-oncology to characterise features of the tumour microenvironment (TME) and correlate them with clinical parameters to guide prognostication and therapy. However, mIHC/IF can be applied to a wide range of organisms in any physiological or disease context. Recent innovation has extended the number of markers that can be detected using slide scanners well beyond the 3-4 markers typically reported in traditional fluorescence microscopy. However, these methods often require sequential antibody staining and stripping, and are not compatible with frozen tissue sections. Using fluorophore-conjugated antibodies, we have established a simple mIHC/IF imaging workflow that enables simultaneous staining and detection of seven markers in a single section of frozen tissue. Coupled with automated whole slide imaging and digital quantification, our data efficiently revealed the tumour-immune complexity in metastatic melanoma. Computational image analysis quantified the immune and stromal cell populations present in the TME as well as their spatial interactions. This imaging workflow can also be performed with an indirect labelling panel consisting of primary and secondary antibodies. Our new methods, combined with digital quantification, will provide a valuable tool for high-quality mIHC/IF assays in immuno-oncology research and other translational studies, especially in circumstances where frozen sections are required for detection of particular markers, or for applications where frozen sections may be preferred, such as spatial transcriptomics.

Intratumoural administration of an NKT cell agonist with CpG promotes NKT cell infiltration associated with an enhanced antitumour response and abscopal effect.

Intratumoural administration of unmethylated cytosine-phosphate-guanine motifs (CpG) to stimulate toll-like receptor (TLR)-9 has been shown to induce tumour regression in preclinical studies and some efficacy in the clinic. Because activated natural killer T (NKT) cells can cooperate with pattern-recognition via TLRs to improve adaptive immune responses, we assessed the impact of combining a repeated dosing regimen of intratumoural CpG with a single intratumoural dose of the NKT cell agonist α-galactosylceramide (α-GalCer). The combination was superior to CpG alone at inducing regression of established tumours in several murine tumour models, primarily mediated by CD8+ T cells. An antitumour effect on distant untreated tumours (abscopal effect) was reliant on sustained activity of NKT cells and was associated with infiltration of KLRG1+ NKT cells in tumours and draining lymph nodes at both injected and untreated distant sites. Cytometric analysis pointed to increased exposure to type I interferon (IFN) affecting many immune cell types in the tumour and lymphoid organs. Accordingly, antitumour activity was lost in animals in which dendritic cells (DCs) were incapable of signaling through the type I IFN receptor. Studies in conditional ablation models showed that conventional type 1 DCs and plasmacytoid DCs were required for the response. In tumour models where the combined treatment was less effective, the addition of tumour-antigen derived peptide, preferably conjugated to α-GalCer, significantly enhanced the antitumour response. The combination of TLR ligation, NKT cell agonism, and peptide delivery could therefore be adapted to induce responses to both known and unknown antigens.

Migratory cues controlling B-lymphocyte trafficking in human lymph nodes.

B-cell migration within lymph nodes (LNs) is crucial to adaptive immune responses. Chemotactic gradients are proposed to drive migration of B cells into follicles, followed by their relocation to specific zones of the follicle during activation, and ultimately egress. However, the molecular drivers of these processes and the cells generating chemotactic signals that affect B cells in human LNs are not well understood. We used immunofluorescence microscopy, flow cytometry and functional assays to study molecular mechanisms of B-cell migration within human LNs, and found subtle but important differences to previous murine models. In human LNs we find CXCL13 is prominently expressed at the follicular edge, often associated with fibroblastic reticular cells located in these areas, whereas follicular dendritic cells show minimal contribution to CXCL13 expression. Human B cells rapidly downregulate CXCR5 on encountering CXCL13, but recover CXCR5 expression in the CXCL13-low environment. These data suggest that the CXCL13 gradient in human LNs is likely to be different from that proposed in mice. We also identify CD68+ CD11c+ PU.1+ tingible body macrophages within both primary and secondary follicles as likely drivers of the sphingosine-1-phosphate (S1P) gradient that mediates B-cell egress from LNs, through their expression of the S1P-degrading enzyme, S1P lyase. Based on our findings, we present a model of B-cell migration within human LNs, which has both similarities and interesting differences to that proposed for mice.

Distinctive Subpopulations of Stromal Cells Are Present in Human Lymph Nodes Infiltrated with Melanoma.

Metastasis of human tumors to lymph nodes (LN) is a universally negative prognostic factor. LN stromal cells (SC) play a crucial role in enabling T-cell responses, and because tumor metastases modulate their structure and function, this interaction may suppress immune responses to tumor antigens. The SC subpopulations that respond to infiltration of malignant cells into human LNs have not been defined. Here, we identify distinctive subpopulations of CD90+ SCs present in melanoma-infiltrated LNs and compare them with their counterparts in normal LNs. The first population (CD90+ podoplanin+ CD105+ CD146+ CD271+ VCAM-1+ ICAM-1+ α-SMA+) corresponds to fibroblastic reticular cells that express various T-cell modulating cytokines, chemokines, and adhesion molecules. The second (CD90+ CD34+ CD105+ CD271+) represents a novel population of CD34+ SCs embedded in collagenous structures, such as the capsule and trabeculae, that predominantly produce extracellular matrix. We also demonstrated that these two SC subpopulations are distinct from two subsets of human LN pericytes, CD90+ CD146+ CD36+ NG2- pericytes in the walls of high endothelial venules and other small vessels, and CD90+ CD146+ NG2+ CD36- pericytes in the walls of larger vessels. Distinguishing between these CD90+ SC subpopulations in human LNs allows for further study of their respective impact on T-cell responses to tumor antigens and clinical outcomes.

Human T cell activation induces synaptic translocation and alters expression of the serine protease inhibitor neuroserpin and its target protease.

Contact between T cells and APCs and activation of an effective immune response trigger cellular polarization and the formation of a structured interface known as the immunological synapse. Interactions across the synapse and secretion of T cell and APC-derived factors into the perisynaptic compartment regulate synapse formation and activation of T cells. We report that the serine protease inhibitor neuroserpin, an axonally secreted protein thought to play roles in the formation of the neuronal synapse and refinement of synaptic activity, is expressed in human naïve effector memory and central memory subsets of CD4(+) and CD8(+) T cells, as well as monocytes, B cells, and NK cells. Neuroserpin partially colocalized with a TGN38/LFA-1-positive vesicle population in T cells and translocates to the immunological synapse upon activation with TCR antibodies or antigen-pulsed APCs. Activation of T cells triggered neuroserpin secretion, a rapid, 8.4-fold up-regulation of the serine protease tissue plasminogen activator, the protease target for neuroserpin, and a delayed, 6.25-fold down-regulation of neuroserpin expression. Evidence of polarization and regulated neuroserpin expression was also seen in ex vivo analyses of human lymph nodes and blood-derived T cells. Increased neuroserpin expression was seen in clusters of T cells in the paracortex of human lymph nodes, with some showing polarization to areas of cell:cell interaction. Our results support a role for neuroserpin and tissue plasminogen activator in activation-controlled proteolytic cleavage of proteins in the synaptic or perisynaptic space to modulate immune cell function.

Identification of germinal centres in the lymph node of a patient with hyperimmunoglobulin M syndrome associated with congenital rubella.

BACKGROUND: The hyper immunoglobulin M syndrome (HIM) associated with congenital rubella infection (rHIM) is an extremely rare disorder, where patients have elevated serum IgM in association with reduced IgG and IgA. We have previously shown that in contrast to X-linked HIM (XHIM), a patient with well-characterised rHIM is able to express functional CD40 ligand, undergo immunoglobulin isotype switching and to generate memory B cells. Here we describe the ultrastructural features of an excised lymph node from this patient. METHODS: An inguinal lymph node was surgically removed and examined histologically as well as by immunohistochemistry. It was then stained with multiple fluorescent dyes to visualize the cellular interactions within the node. Flow cytometry was undertaken on a cellular suspension from the node. FINDINGS: Our patient has normal lymph node architecture by light microscopy. Immunohistochemistry studies showed the presence of scattered germinal centres. Polychromatic immunofluorescence staining showed disruption of the architecture with mostly abnormal germinal centres. A small number of relatively intact germinal centres were identified. Both IgM and IgG bearing cells were identified in germinal centres. INTERPRETATION: In contrast to XHIM where germinal centres are absent, the presence of small numbers of relatively normal germinal centres explain our previous identification of isotype switched memory B cells in rHIM.

Sphingosine-1-phosphate lyase is expressed by CD68+ cells on the parenchymal side of marginal reticular cells in human lymph nodes.

Lymph nodes (LNs) form the intersection between the vascular and lymphatic systems. Lymphocytes and antigen-presenting cells (APCs) traffic between these systems, but the barriers crossed during this trafficking in human LNs are poorly defined. We identified a population of cells in human LNs that lines the boundary between the parenchyma and lymphatic sinuses, consistent with descriptions of marginal reticular cells (MRCs) in murine LNs. Human MRCs are CD141(high) podoplanin(+), CD90(+), ICAM1(+), and VCAM1(+) but lack endothelial and hematopoietic cell markers, or alpha-smooth muscle actin. We then examined expression of the enzyme sphingosine-1-phosphate (S1P) lyase (SGPL1) relative to the boundary defined by MRCs. SGPL1 expression was almost exclusively restricted to cells on the parenchymal side of MRCs, consistent with a role in maintaining the S1P gradient between the sinuses and the parenchyma. Surprisingly the cells expressing SGPL1 in the parenchyma were CD68(+) APCs. CD68(+) APCs generated from human monocytes were able to internalize and irreversibly degrade S1P, and this activity was inhibited by the S1P analogue FTY720. This work provides a map of the key structures at the boundary where human lymphocytes egress into sinuses, and identifies a novel potential mechanism for the activity of S1P analogues in humans.

Mapping the distinctive populations of lymphatic endothelial cells in different zones of human lymph nodes.

The lymphatic sinuses in human lymph nodes (LNs) are crucial to LN function yet their structure remains poorly defined. Much of our current knowledge of lymphatic sinuses derives from rodent models, however human LNs differ substantially in their sinus structure, most notably due to the presence of trabeculae and trabecular lymphatic sinuses that rodent LNs lack. Lymphatic sinuses are bounded and traversed by lymphatic endothelial cells (LECs). A better understanding of LECs in human LNs is likely to improve our understanding of the regulation of cell trafficking within LNs, now an important therapeutic target, as well as disease processes that involve lymphatic sinuses. We therefore sought to map all the LECs within human LNs using multicolor immunofluorescence microscopy to visualize the distribution of a range of putative markers. PROX1 was the only marker that uniquely identified the LECs lining and traversing all the sinuses in human LNs. In contrast, LYVE1 and STAB2 were only expressed by LECs in the paracortical and medullary sinuses in the vast majority of LNs studied, whilst the subcapsular and trabecular sinuses lacked these molecules. These data highlight the existence of at least two distinctive populations of LECs within human LNs. Of the other LEC markers, we confirmed VEGFR3 was not specific for LECs, and CD144 and CD31 stained both LECs and blood vascular endothelial cells (BECs); in contrast, CD59 and CD105 stained BECs but not LECs. We also showed that antigen-presenting cells (APCs) in the sinuses could be clearly distinguished from LECs by their expression of CD169, and their lack of expression of PROX1 and STAB2, or endothelial markers such as CD144. However, both LECs and sinus APCs were stained with DCN46, an antibody commonly used to detect CD209.

Characterization of mesenchymal progenitor cell populations directly derived from human dermis.

Mesenchymal stem cell (MSC) and progenitor cell (MPC) populations in human dermis remain poorly characterized, despite their importance to wound repair and the pathogenesis of many skin diseases. To identify MSC/MPC populations in human dermis we developed an 11-marker flow cytometry technique that enabled sorting of mesenchymal cell populations for functional assays, using adipose-derived stem cells (ASCs) from human adipose tissue as a positive control. Two populations of dermal cells had similar phenotypes to ASCs: both were CD34(+) CD73(+) CD105(-)/low, and lacked expression of c-kit (CD117) and hematopoietic or vascular markers (CD31, CD45, CD146, and HLA-DR). However, whereas ASCs were CD36(+/-) CD90(+), dermal mesenchymal progenitor cells (DMPCs) were split between a dominant CD36(-) CD90(+) population (DMPC1) and a small CD36(+) CD90(-) population (DMPC2). Both these populations were capable of differentiating into adipocytes, but only DMPC1 localized to a perivascular location, similar to that reported for ASCs. Re-gating of the flow cytometry data revealed that both DMPC1 and DMPC2 were part of CD45(-) CD73(+) CD146(-) populations with variable expression of CD34. This suggests that CD34 may not be a stable marker of DMPC populations in human dermis, consistent with data from MSCs in human bone marrow, and with the loss of CD34 we observed from both ASCs and DMPCs on cell culture. These data enable future study of DMPCs in health and disease, and may also explain why some mesenchymal cell lines derived from human dermis exhibit characteristics of MSCs.

The interaction between bacteria and mucosal immunity in chronic rhinosinusitis: a prospective cross-sectional analysis.

BACKGROUND: We have detected intramucosal bacteria within the sinus mucosa of patients with chronic rhinosinusitis (CRS), but our attempts at characterizing these did not yield any discernible genotypic or phenotypic differences from surface bacteria. We hypothesized that the presence of intramucosal microcolonies reflected host mucosal immune dysfunction. This study characterizes the activation status of T cells, B cells, and macrophages in the sinus mucosa of patients with CRS and controls and determines the impact of bacteria on mucosal immunology. METHODS: Swabs and mucosal biopsy specimens were taken from 27 patients with CRS undergoing sinus surgery and 9 patients with normal sinuses having transnasal pituitary surgery. Microcolonies were detected using Gram staining, and the immune cells were characterized by immunohistochemical techniques. RESULTS: Swab culture rates for Staphylococcus aureus were similar between CRS and controls. However, there were significantly more intramucosal microcolonies in CRS (59% versus 11%) than in controls (p = 0.02). There were significantly more immune cells in CRS. Percentage of activated T and B cells were similar between CRS and controls, but there were significantly more CD163(+) M2 macrophages in patients with CRS (p = 0.0004). Furthermore, percentage of CD163(+) macrophages showed a positive correlation with disease severity. The presence of bacteria had no impact on immunology or disease severity. CONCLUSION: Tolerance of intramucosal microcolonies in CRS may reflect altered macrophage function in the host mucosa. The clinical severity of CRS is also dependent on the host mucosa immune dysfunction, rather than the presence of intramucosal microcolonies.

The interaction between bacteria and mucosal immunity in chronic rhinosinusitis: A prospective cross-sectional analysis.

BACKGROUND: We have detected intramucosal bacteria within the sinus mucosa of patients with chronic rhinosinusitis (CRS), but our attempts at characterizing these did not yield any discernible genotypic or phenotypic differences from surface bacteria. We hypothesized that the presence of intramucosal microcolonies reflected host mucosal immune dysfunction. This study characterizes the activation status of T cells, B cells, and macrophages in the sinus mucosa of patients with CRS and controls and determines the impact of bacteria on mucosal immunology. METHODS: Swabs and mucosal biopsy specimens were taken from 27 patients with CRS undergoing sinus surgery and 9 patients with normal sinuses having transnasal pituitary surgery. Microcolonies were detected using Gram staining, and the immune cells were characterized by immunohistochemical techniques. RESULTS: Swab culture rates for Staphylococcus aureus were similar between CRS and controls. However, there were significantly more intramucosal microcolonies in CRS (59% versus 11%) than in controls (p = 0.02). There were significantly more immune cells in CRS. Percentage of activated T and B cells were similar between CRS and controls, but there were significantly more CD163+ M2 macrophages in patients with CRS (p = 0.0004). Furthermore, percentage of CD163+ macrophages showed a positive correlation with disease severity. The presence of bacteria had no impact on immunology or disease severity. CONCLUSION: Tolerance of intramucosal microcolonies in CRS may reflect altered macrophage function in the host mucosa. The clinical severity of CRS is also dependent on the host mucosa immune dysfunction, rather than the presence of intramucosal microcolonies.

Human CD141+ (BDCA-3)+ dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens.

The characterization of human dendritic cell (DC) subsets is essential for the design of new vaccines. We report the first detailed functional analysis of the human CD141+ DC subset. CD141+ DCs are found in human lymph nodes, bone marrow, tonsil, and blood, and the latter proved to be the best source of highly purified cells for functional analysis. They are characterized by high expression of toll-like receptor 3, production of IL-12p70 and IFN-beta, and superior capacity to induce T helper 1 cell responses, when compared with the more commonly studied CD1c+ DC subset. Polyinosine-polycytidylic acid (poly I:C)-activated CD141+ DCs have a superior capacity to cross-present soluble protein antigen (Ag) to CD8+ cytotoxic T lymphocytes than poly I:C-activated CD1c+ DCs. Importantly, CD141+ DCs, but not CD1c+ DCs, were endowed with the capacity to cross-present viral Ag after their uptake of necrotic virus-infected cells. These findings establish the CD141+ DC subset as an important functionally distinct human DC subtype with characteristics similar to those of the mouse CD8alpha+ DC subset. The data demonstrate a role for CD141+ DCs in the induction of cytotoxic T lymphocyte responses and suggest that they may be the most relevant targets for vaccination against cancers, viruses, and other pathogens.

Neutrophil influx and chemokine production during the early phases of the antitumor response to the vascular disrupting agent DMXAA (ASA404).

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) acts through tumor vascular disruption and cytokine production and is the first of its class to enter phase 3 trials. We characterized leukocytes and cytokines in murine Colon 38 tumors before and after DMXAA treatment. Tumor mass declined 50% 24 hours after DMXAA administration, but the leukocyte count per gram of tumor increased threefold owing to a large influx of Ly6G(+)CD11b(+)F4/80(-) cells with the morphology of neutrophils. However, B and T lymphocytes, natural killer cells, and macrophages in the tumor all decreased in numbers. Seven chemokines were substantially induced in the tumor, spleen, and serum 4 hours after DMXAA administration. Using cultured spleen cell subpopulations, CD11b(+) cells (largely monocytes and macrophages) were shown to be the primary producers of tumor necrosis factor alpha, interleukin 6 (IL-6), and macrophage inflammatory 1alpha (MIP-1alpha). CD49b(+) natural killer cells produced IP-10, whereas CD45R(+) B lymphocytes produced regulated upon activation normal T cell express sequence. T lymphocytes were not major producers of cytokines in the response to DMXAA. Murine peripheral blood leukocytes (PBLs) produced a similar panel of cytokines in culture to that detected in mouse serum after DMXAA treatment. Cytokines in human PBL cultures were subsequently measured with the aim of identifying potential serum markers of the human response to DMXAA. IP-10 (P < .001), monocyte chemoattractant protein 1 (P < .001), and sCD40L (P < .01) were decreased, whereas IL-8 (P < .001) and MIP-1alpha (P = .03) were increased in DMXAA-treated compared with untreated PBL cultures from a group of 12 donors.

Distinctive localization of antigen-presenting cells in human lymph nodes.

Professional antigen-presenting cells (APCs) are sentinel cells of the immune system that present antigen to T lymphocytes and mediate an appropriate immune response. It is therefore surprising that knowledge of the professional APCs in human lymph nodes is limited. Using 3-color immunohistochemistry, we have identified APCs in human lymph nodes, excluding plasmacytoid APCs, that fall into 2 nonoverlapping classes: (1) CD209+ APCs, coexpressing combinations of CD206, CD14, and CD68, that occupied the medullary cords, lined the capsule and trabeculae and were also scattered throughout the diffuse T-lymphocyte areas of the paracortex; and (2) APCs expressing combinations of CD1a, CD207, and CD208, that were always restricted to the paracortex. Surprisingly, this second class of APCs was almost entirely absent from many lymph nodes. Our data suggest that most CD208+ cells, often referred to as "interdigitating cells," derive from migratory APCs, and that the major APC subset consistently resident in the paracortex of human lymph nodes is the CD209+ subset. All APC subsets were demonstrated to be in close contact with the fibroreticular network. The identification of 2 distinct APC populations in the paracortex of human lymph nodes has important implications for understanding T-lymphocyte responses and optimizing vaccine design.

CD14+ antigen-presenting cells in human dermis are less mature than their CD1a+ counterparts.

We recently demonstrated that three antigen-presenting cell (APC) subsets exist in the healthy human dermis, CD14(+) and CD1a(+) dermal APCs and migratory dermal Langerhans cells. Here, we extend these findings by defining CD208 as an exclusive marker of migratory dermal Langerhans cells, confirming that migratory dermal Langerhans cells (CD1a(high) CD207(+) CD208(+)) and CD1a(+) dermal APCs (CD1a(mid) CD207(-) CD208(-)) are two distinct APC populations. Using flow cytometry and multicolor fluorescence immunohistochemistry, we demonstrated that there were striking differences between CD1a(+) and CD14(+) dermal APCs in their expression of pattern recognition receptors and maturation markers. Expression of Toll-like receptor (TLR) 2, CD206 and CD209 was largely restricted to CD14(+) dermal APCs. Consistent with these observations, most CD14(+) dermal APCs expressed an immature phenotype when compared with CD1a(+) dermal APCs, which expressed high levels of the maturation marker CD83 on their cell surface. However, a subset of CD14(+) dermal APCs also expressed cell-surface CD83, associated with a loss of cell-surface TLR2, suggesting that they have the capacity to mature. CD14(+) dermal APCs are therefore the dominant cutaneous APC population capable of sensing ligands recognized by CD206, CD209 and TLR2 and subsequently may have the potential to mature. CD68 expression was largely restricted to a subset of CD14(+) dermal APCs, while both CD14(+) and CD1a(+) dermal APCs expressed CD11b and CD11c. These findings have important implications for understanding cutaneous immune responses in humans and for the optimization of vaccine delivery via the skin.