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Highlights d Caveolae influence contractile tension in epithelial monolayers d Caveolin-1 depletion enhances epithelial tension via PtdIns(4,5)P 2 signaling d Elevated epithelial tension inhibits oncogenic cell extrusion

Cross-presentation is the mechanism by which exogenous Ag is processed for recognition by CD8 + T cells. Murine CD8α + DCs are specialized at cross-presenting soluble and cellular Ag, but in humans this process is poorly characterized. In this study, we examined uptake and cross-presentation of soluble and cellular Ag by human blood CD141 + DCs, the human equivalent of mouse CD8α + DCs, and compared them with human monocyte-derived DCs (MoDCs) and blood CD1c + DC subsets. MoDCs were superior in their capacity to internalize and cross-present soluble protein whereas CD141 + DCs were more efficient at ingesting and cross-presenting cellular Ag. Whilst cross-presentation by CD1c + DCs and CD141 + DCs was dependent on the proteasome, and hence cytosolic translocation, cross-presentation by MoDCs was not. Inhibition of endosomal acidification enhanced cross-presentation by CD1c + DCs and MoDCs but not by CD141 + DCs. These data demonstrate that CD1c + DCs, CD141 + DCs, and MoDCs are capable of crosspresentation; however, they do so via different mechanisms. Moreover, they demonstrate that human CD141 + DCs, like their murine CD8α + DC counterparts, are specialized at cross-presenting cellular Ag, most likely mediated by an enhanced capacity to ingest cellular Ag combined with subtle changes in lysosomal pH during Ag processing and use of the cytosolic pathway.Keywords: Antigen processing r Cross-presentation r Human dendritic cells Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionDendritic cells (DCs) are professional APCs that are uniquely able to process and present antigen (Ag) to prime naïve T-cell Correspondence: Dr. Kristen J. Radford e-mail: kristen.radford@mater.uq.edu.au responses. DCs in human and mouse can be classified into a number of subsets that vary in location, phenotype, and specialized function [1]. These include (i) inflammatory monocyte-derived DCs (MoDCs) that develop from monocytes and are rapidly * These authors contributed equally to this work.C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 330 Meng-Chieh Chiang et al. Eur. J. Immunol. 2016. 46: 329-339 recruited to sites of inflammation, (ii) plasmacytoid DCs, which are key producers of type I IFN, and (iii) "classical" or "conventional" DCs (cDCs), which can be further categorized based on location into "lymphoid-resident" and "migratory" DCs [1]. The lymphoidresident DCs capture Ag directly in situ, whereas migratory DCs reside in the peripheral organs (e.g. lung, skin, and gut) where they capture Ag then migrate to lymphoid tissues to share their Ag with other lymphoid-resident DCs, or present Ag directly to T cells. cDCs can be further segregated into two main subsets: (i) the mouse CD11b + cDC subset and human CD1c + DC equivalent; and (ii) the mouse CD8α + lymphoid-resident DC, related mouse CD103 + tissue resident cDCs, and the human equivalent CD141 + DC that can now be collectively defined by coexpression of the C-type lectin-like re...

BackgroundThe conventional type 1 dendritic cell subset (cDC1) is indispensable for tumor immune responses and the efficacy of immune checkpoint inhibitor (ICI) therapies in animal models but little is known about the role of the human CD141+ DC cDC1 equivalent in patients with melanoma.MethodsWe developed a flow cytometry assay to quantify and characterize human blood DC subsets in healthy donors and patients with stage 3 and stage 4 metastatic melanoma. To examine whether harnessing CD141+ DCs could improve responses to ICIs in human melanoma, we developed a humanized mouse model by engrafting immunodeficient NSG-SGM3 mice with human CD34+ hematopoietic stem cells (HSCs) from umbilical cord blood followed by transplantation of a human melanoma cell line and treatment with anti-programmed cell death protein-1 (anti-PD-1).ResultsBlood CD141+ DC numbers were significantly reduced in patients with stage 4 melanoma compared with healthy controls. Moreover, CD141+ DCs in patients with melanoma were selectively impaired in their ability to upregulate CD83 expression after stimulation with toll-like receptor 3 (TLR3) and TLR7/8 agonists ex vivo. Although DC numbers did not correlate with responses to anti-PD-1 and/or anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) ICIs, their numbers and capacity to upregulate CD83 declined further during treatment in non-responding patients. Treatment with anti-PD-1 was ineffective at controlling tumor growth in humanized mice but efficacy was enhanced by indirectly expanding and activating DCs in vivo with fms-like tyrosine kinase-3 ligand (Flt3L) and a TLR3 agonist. Moreover, intratumoral injections of CD141+ DCs resulted in reduced tumor growth when combined with anti-PD-1 treatment.ConclusionsThese data illustrate quantitative and qualitative impairments in circulating CD141+ DCs in patients with advanced melanoma and that increasing CD141+ DC number and function is an attractive strategy to enhance immunogenicity and response rates to ICIs.

Tumours most often arise from progression of precursor clones within a single anatomical niche. In the bone marrow, clonal progenitors can undergo malignant transformation to acute leukaemia, or differentiate into immune cells that contribute to disease pathology in peripheral tissues1–4. Outside the marrow, these clones are potentially exposed to a variety of tissue-specific mutational processes, although the consequences of this are unclear. Here we investigate the development of blastic plasmacytoid dendritic cell neoplasm (BPDCN)—an unusual form of acute leukaemia that often presents with malignant cells isolated to the skin5. Using tumour phylogenomics and single-cell transcriptomics with genotyping, we find that BPDCN arises from clonal (premalignant) haematopoietic precursors in the bone marrow. We observe that BPDCN skin tumours first develop at sun-exposed anatomical sites and are distinguished by clonally expanded mutations induced by ultraviolet (UV) radiation. A reconstruction of tumour phylogenies reveals that UV damage can precede the acquisition of alterations associated with malignant transformation, implicating sun exposure of plasmacytoid dendritic cells or committed precursors during BPDCN pathogenesis. Functionally, we find that loss-of-function mutations in Tet2, the most common premalignant alteration in BPDCN, confer resistance to UV-induced cell death in plasmacytoid, but not conventional, dendritic cells, suggesting a context-dependent tumour-suppressive role for TET2. These findings demonstrate how tissue-specific environmental exposures at distant anatomical sites can shape the evolution of premalignant clones to disseminated cancer.

Dendritic cells (DCs) as professional antigen-presenting cells with unique T-cell stimulatory capacity represent a potential means by which to improve response rates towards immune checkpoint inhibitor antibodies (ICIs) such as anti-PD-1 (pembrolizumab) in melanoma. The conventional type 1 DC subset (cDC1) is indispensable for the efficacy of ICIs in animal models; however, very little is known about the role of cDC1 in human cancer patients. To address this, we developed a whole-blood assay for quantifying and characterizing human DC subsets (cDC1, cDC2 and plasmacytoid DCs [pDCs]) by flow cytometry and compared these in healthy donors and stage III and IV metastatic melanoma patients. cDC1 and pDC numbers were significantly reduced in stage IV melanoma patients compared to healthy controls. Moreover, cDC1s in melanoma patients were selectively impaired in their ability to upregulate CD83 expression after stimulation with TLR3 and TLR7/8 agonists ex vivo. Although DC numbers did not correlate with responses to anti-PD-1 and/or anti-CTLA-4 ICIs, their numbers and capacity to upregulate CD83 declined further during treatment in nonresponding patients. To examine whether harnessing cDC1 could improve responses to ICIs in human melanoma, we developed a humanized mouse model by engrafting immunodeficient NSG-SGM3 mice with CD34+ hematopoietic stem cells from umbilical cord blood followed by transplantation of a human melanoma cell line. Treatment with anti-PD-1 in this model was ineffective at controlling tumor growth, but efficacy was enhanced by indirectly expanding and activating DCs in vivo with Flt-3 ligand (Flt3L) and TLR3 agonist polyI:C. Moreover, intratumoral injection of cDC1s, but not cDC2s, resulted in reduced tumor growth when combined with anti-PD-1 treatment. Together, these data illustrate impairments in cDC1 in advanced melanoma patients and provide rationale for harnessing them to increase immunogenicity and response rates to ICIs. Citation Format: Liam O’Brien, Yoke Seng Lee, Carina Walpole, Ingrid Leal Rojas, Kelly-Anne Masterman, Victoria Atkinson, Andrew Barbour, Kristen Radford. Human CD141+ dendritic cells (cDC1) are impaired in advanced melanoma patients but can be targeted to increase efficacy of anti-PD-1 checkpoint inhibitor therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B42.

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