Ontogenic, phenotypic and functional characterization of dendritic cells and macrophages in skin squamous cell carcinoma

Les carcinomes épidermoïdes cutanés (CEC) sont des cancers invasifs du kératinocyte qui se développent suite à la subversion du système immunitaire. Une reprogrammation de l’immunité antitumorale par stimulation locale des cellules dendritiques (DC) et des macrophages pourrait être bénéfique. Dans ce contexte, l’objectif de ma thèse a été de comprendre l’hétérogénéité ontogénique, phénotypique et fonctionnelle de ces cellules immunitaires au cours de la carcinogenèse cutanée afin de développer des approches thérapeutiques les stimulant. Nous avons, d’abord, caractérisé les macrophages de la peau de souris et infiltrant les CEC chez l’homme. Nous avons identifié par cytométrie en flux spectrale une population de macrophages matures, autofluorescents et résidents de la peau de souris. Ces macrophages résidents présentent une polarisation fonctionnelle d’homéostasie et de réparation tissulaire. Dans les CEC humains, les macrophages autofluorescents semblent avoir les mêmes caractéristiques que leurs équivalents dans la peau de souris. Dans une seconde étude, nous avons mis en évidence que les DC associées aux tumeurs sont dysfonctionnelles. Une immunothérapie locale composée d’un agoniste de TLR9 et d’un anticorps bloquant la signalisation sous le récepteur à l’IL-10 induit la régression de tumeurs cutanées. Cette approche permet la reprogrammation fonctionnelle des DC et la génération de lymphocytes T CD8+ producteurs d’IFNγ, de TNFα et d’IL-17. Ces résultats mettent en évidence l’hétérogénéité fonctionnelle des cellules myéloïdes dans la peau et les CEC. Leur reprogrammation fonctionnelle permettrait le développement de nouvelles thérapeutiques contre les CEC chez l’homme.Skin Squamous cell carcinoma (sSCC) are invasive keratinocyte tumor that develop after immune system subversion. The reprogrammation of anti-tumoral immunity using local stimulation of dendritic cells (DC) and macrophages could be useful. In this line, the aim of my thesis was to understand ontogenic, phenotypic and functional heterogeneity of these cell subsets along skin carcinogenesis to develop new immunotherapies. First, we characterized skin macrophage subsets in mouse and those infiltrating sSCC in human. Using spectral flow cytometry, we identified matured autofluorescent tissue-resident macrophages. These macrophages are anti-inflammatory polarized. In human sSCC, autofluorescent macrophages seem to have same properties that their mouse counterparts. In second study, we identified tumor-infiltrating DC with altered functions. We used a local immunotherapy composed by a TLR9 agonist and blocking antibody against α-chain of IL-10 receptor. This combination induced tumor regression through DC reprogrammation and IFNγ+, TNFα+ IL17A+ T CD8+ lymphocyte generation. These results highlight functional myeloid heterogeneity in skin and sSCC. Their reprogrammation could promote the development of immunotherapies against sSCC in human

Engineered niches support the development of human dendritic cells in humanized mice

International audienceClassical dendritic cells (cDCs) are rare sentinel cells specialized in the regulation of adaptive immunity. Modeling cDC development is crucial to study cDCs and harness their therapeutic potential. Here we address whether cDCs could differentiate in response to trophic cues delivered by mesenchymal components of the hematopoietic niche. We find that mesenchymal stromal cells engineered to express membrane-bound FLT3L and stem cell factor (SCF) together with CXCL12 induce the specification of human cDCs from CD34+ hematopoietic stem and progenitor cells (HSPCs). Engraftment of engineered mesenchymal stromal cells (eMSCs) together with CD34+ HSPCs creates an in vivo synthetic niche in the dermis of immunodeficient mice driving the differentiation of cDCs and CD123+AXL+CD327+ pre/AS-DCs. cDC2s generated in vivo display higher levels of resemblance with human blood cDCs unattained by in vitro-generated subsets. Altogether, eMSCs provide a unique platform recapitulating the full spectrum of cDC subsets enabling their functional characterization in vivo

Que la lumière soit. Et si ce n’était plus seulement vrai !

International audienceThe last decade has been an era of accelerated technological progress for flow cytometry. New technologies have been developed such as mass cytometry in which standard fluorochromes have been replaced by lanthanide-based non-radioactive metals and by spectral cytometry that measures the complete fluorescence spectrum. In this review, we schematically describe conventional, mass and spectral cytometry and present the plus and minus of each technology

NKp46+ Innate Lymphoid Cells Dampen Vaginal CD8 T Cell Responses following Local Immunization with a Cholera Toxin-Based Vaccine

International audienceInnate and adaptive immune cells work in concert to generate efficient protection at mucosal surface. Vaginal mucosa is an epithelial tissue that contains innate and adaptive immune effector cells. Our previous studies demonstrated that vaginal administration of Cholera toxin -based vaccines generate antigen-specific CD8 T cells through the stimulation of local dendritic cells (DC). Innate lymphoid cells (ILC) are a group of lymphocytes localized in epithelial tissues that have important immune functions against pathogens and in tissue homeostasis. Their contribution to vaccine-induced mucosal T cell responses is an important issue for the design of protective vaccines. We report here that the vaginal mucosa contains a heterogeneous population of NKp46+ ILC that includes conventional NK cells and ILC1-like cells. We show that vaginal NKp46+ ILC dampen vaccine-induced CD8 T cell responses generated after local immunization. Indeed, in vivo depletion of NKp46+ ILC with anti-NK1.1 antibody or NKG2D blockade increases the magnitude of vaginal OVA-specific CD8 T cells. Furthermore, such treatments also increase the number of DC in the vagina. NKG2D ligands being expressed by vaginal DC but not by CD8 T cells, these results support that NKp46+ ILC limit mucosal CD8 T cell responses indirectly through the NKG2D-dependent elimination of vaginal DC. Our data reveal an unappreciated role of NKp46+ ILC in the regulation of mucosal CD8 T cell responses

Autofluorescence identifies highly phagocytic tissue-resident macrophages in mouse and human skin and cutaneous squamous cell carcinoma

International audienceMacrophages from human and mouse skin share phenotypic and functional features, but remain to be characterized in pathological skin conditions. Skinresident macrophages are known to derive from embryonic precursors or from adult hematopoiesis. In this report, we investigated the origins, phenotypes and functions of macrophage subsets in mouse and human skin and in cutaneous squamous cell carcinoma (cSCC) using the spectral flow cytometry technology that enables cell autofluorescence to be considered as a full-fledged parameter. Autofluorescence identifies macrophage subsets expressing the CD206 mannose receptor in human peri-tumoral skin and cSCC. In mouse, all AF + macrophages express the CD206 marker, a subset of which also displaying the TIM-4 marker. While TIM-4-CD206 + AF + macrophages can differentiate from bone-marrow monocytes and infiltrate skin and tumor, TIM-4 identifies exclusively a skin-resident AF + macrophage subset that can derive from prenatal hematopoiesis which is absent in tumor core. In mouse and human, AF + macrophages from perilesional skin and cSCC are highly phagocytic cells contrary to their AF-counterpart, thus identifying autofluorescence as a bona fide marker for phagocytosis. Our data bring to light autofluorescence as a functional marker characterizing subsets of phagocytic macrophages in skin and cSCC. Autofluorescence can thus be considered as an attractive marker of function of macrophage subsets in pathological context

Tumor-Associated Neutrophils Dampen Adaptive Immunity and Promote Cutaneous Squamous Cell Carcinoma Development

Cutaneous squamous cell carcinoma (cSCC) development has been linked to immune dysfunctions but the mechanisms are still unclear. Here, we report a progressive infiltration of tumor-associated neutrophils (TANs) in precancerous and established cSCC lesions from chemically induced skin carcinogenesis. Comparative in-depth gene expression analyses identified a predominant protumor gene expression signature of TANs in lesions compared to their respective surrounding skin. In addition, in vivo depletion of neutrophils delayed tumor growth and significantly increased the frequency of proliferating IFN-γ (interferon-γ)-producing CD8+ T cells. Mechanisms that limited antitumor responses involved high arginase activity, production of reactive oxygen species (ROS) and nitrite (NO), and the expression of programmed death-ligand 1 (PD-L1) on TAN, concomitantly with an induction of PD-1 on CD8+ T cells, which correlated with tumor size. Our data highlight the relevance of targeting neutrophils and PD-L1-PD-1 (programmed death-1) interaction in the treatment of cSCC

Functional properties of vaginal Eomes⁺ NK cells and Eomes^- ILC1-like cells.

<p>(A) Bidimensional dot plots (left panel) show expression of CD27 and CD11b on gated Eomes⁺ NKp46⁺ CD3^- cells from spleen, GLN and vaginas of naive C57BL/6 mice. Numbers in quadrants represent the percentage of cells for each subset. Histograms bars (right panel) represent results from five independent experiments expressed as mean values + SEM, <i>n</i> = 10 mice. <i>**p<0</i>.<i>01</i>, <i>***p<0</i>.<i>001</i>, <i>****p<0</i>.<i>0001</i> Mann-Whitney <i>U</i> test. Spl.: spleen; GLN: genital lymph nodes; Vag.: vagina. Frequencies of CD107a⁺ and IFN-γ⁺ producing Eomes⁺ NK cells (B) and Eomes^- ILC1-like cells (C) in cell suspensions from spleen, GLN and vagina of naive C57BL/6 mice. Cells (0.5 million/well) were stimulated <i>in vitro</i> for 4 hours with plate-bound isotype control (IC), anti NK1.1 mAb, PMA/ionomycin, IL-12 + IL-18 cytokines or YAC-1 tumor cells. CD107a and IFN-γ expression on cells were assessed by flow cytometry. Histogram bars represent results from three independent experiments expressed as mean percentages + SEM, <i>n</i> = 5 mice. <i>**p<0</i>.<i>01</i>, <i>***p<0</i>.<i>001</i>, Mann-Whitney <i>U</i> test. (D) Bidimensional dot plots (left panel) show expression of TNFα and TRAIL on gated Eomes^- ILC1 and Eomes⁺ NK cells from liver and vaginas of naive C57BL/6 mice after stimulation <i>in vitro</i> for 4 hours with PMA/ionomycin. Numbers in quadrants represent the percentage of cells for each subset. Results are representative of two independent experiments with n = 4 mice.</p

Dendritic cell-targeted therapy expands CD8 T cell responses to bona-fide neoantigens in lung tumors

International audienceCross-presentation by type 1 DCs (cDC1) is critical to induce and sustain antitumoral CD8 T cell responses to model antigens, in various tumor settings. However, the impact of cross-presenting cDC1 and the potential of DC-based therapies in tumors carrying varied levels of bona-fide neoantigens (neoAgs) remain unclear. Here we develop a hypermutated model of non-small cell lung cancer in female mice, encoding genuine MHC-I neoepitopes to study neoAgs-specific CD8 T cell responses in spontaneous settings and upon Flt3L + αCD40 (DC-therapy). We find that cDC1 are required to generate broad CD8 responses against a range of diverse neoAgs. DC-therapy promotes immunogenicity of weaker neoAgs and strongly inhibits the growth of high tumor-mutational burden (TMB) tumors. In contrast, low TMB tumors respond poorly to DC-therapy, generating mild CD8 T cell responses that are not sufficient to block progression. scRNA transcriptional analysis, immune profiling and functional assays unveil the changes induced by DC-therapy in lung tissues, which comprise accumulation of cDC1 with increased immunostimulatory properties and less exhausted effector CD8 T cells. We conclude that boosting cDC1 activity is critical to broaden the diversity of anti-tumoral CD8 T cell responses and to leverage neoAgs content for therapeutic advantage