Proliferating macrophages in human tumours show characteristics of monocytes responding to myelopoietic growth factors.

Macrophages play essential roles in maintaining tissue homeostasis and immune defence. However, their extensive infiltration into tumours has been linked to adverse outcomes in multiple human cancers. Within the tumour microenvironment (TME), tumour-associated macrophages (TAMs) promote tumour growth and metastasis, making them prime targets for cancer immunotherapy. Recent single-cell analysis suggest that proliferating TAMs accumulate in human cancers, yet their origins and differentiation pathways remain uncertain. Here, we show that a subpopulation of CD163+ TAMs proliferates in situ within the TME of melanoma, lung cancer, and breast cancer. Consistent with their potential role in suppressing anti-tumour activities of T cells, CD163+ TAMs express a range of potent immunosuppressive molecules, including PD-L1, PD-L2, IL-10, and TGF-ß. Other phenotypic markers strongly suggested that these cells originate from CD14+ CCR2+ monocytes, a cell population believed to have minimal capacity for proliferation. However, we demonstrate in vitro that certain myelopoietic cytokines commonly available within the TME induce robust proliferation of human monocytes, especially the combination of interleukin 3 (IL-3) and Macrophage Colony-Stimulating Factor 1 (M-CSF). Monocytic cells cultured with these cytokines efficiently modulate T cell proliferation, and their molecular phenotype recapitulates that of CD163+ TAMs. IL-3-driven proliferation of monocytic cells can be completely blocked by IL-4, associated with the induction of CDKN1A, alongside the upregulation of transcription factors linked to dendritic cell function, such as BATF3 and IRF4. Taken together, our work suggests several novel therapeutic routes to reducing immunosuppressive TAMs in human tumours, from blocking chemokine-mediated recruitment of monocytes to blocking their proliferation.

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.

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.

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.