Over the past decade, immunotherapy has become a powerful and evident tool in the fight against cancers. Notably, the rise of checkpoint blockade using monoclonal antibodies (anti-CTLA4, anti-PD1) to ...avoid interaction between inhibitory molecules allowed the betterment of patient care. Indeed, immunotherapies led to increased overall survival in forms of cutaneous melanoma or lung cancer. However, the percentage of patients responding varies from 20 to 40% depending on the type of cancer and on the expression of the target molecules by the tumor. This is due to the tumor microenvironment which allows the acquisition of resistance mechanisms to immunotherapies by tumor cells. These are closely linked to the architecture and cellular composition of the tumor microenvironment. This one acts on different parameters such as the immune cells infiltrate its composition and therefore, favors the recruitment of immunosuppressive cells as well as the tumor expression of checkpoint inhibitors such as Programmed Death Ligand-1 (PD-L1). Therefore, the analysis and modeling of the complexity of the microenvironment is an important parameter to consider, not only in the search for new therapies but also for the identification and stratification of patients likely to respond to immunotherapy. This is why the use of 3D culture models, reflecting the architecture and cellular composition of a tumor, is essential in immuno-oncology studies. Nowadays, there are several 3-D culture methods such as spheroids and organoids, which are applicable to immuno-oncology. In this review we evaluate 3D culture models as tools for the development of treatments in the field of immuno-oncology.
Endometrial Cancer is the most common cancer in the female genital tract in developed countries, and with its increasing incidence due to risk factors such as aging and obesity tends to become a ...public health issue. However, its immune environment has been less characterized than in other tumors such as breast cancers. NK cells are cytotoxic innate lymphoid cells that are considered as a major anti-tumoral effector cell type which function is drastically altered in tumors which participates to tumor progression. Here we characterize tumor NK cells both phenotypically and functionally in the tumor microenvironment of endometrial cancer. For that, we gathered endometrial tumors, tumor adjacent healthy tissue, blood from matching patients and healthy donor blood to perform comparative analysis of NK cells. First we found that NK cells were impoverished in the tumor infiltrate. We then compared the phenotype of NK cells in the tumor and found that tumor resident CD103
NK cells exhibited more co-inhibitory molecules such as Tigit, and TIM-3 compared to recruited CD103
NK cells and that the expression of these molecules increased with the severity of the disease. We showed that both chemokines (CXCL12, IP-10, and CCL27) and cytokines profiles (IL-1β and IL-6) were altered in the tumor microenvironment and might reduce NK cell function and recruitment to the tumor site. This led to hypothesize that the tumor microenvironment reduces resident NK cells cytotoxicity which we confirmed by measuring cytotoxic effector production and degranulation. Taken together, our results show that the tumor microenvironment reshapes NK cell phenotype and function to promote tumor progression.
Interactions of developing T cells with Aire
medullary thymic epithelial cells expressing high levels of MHCII molecules (mTEC
) are critical for the induction of central tolerance in the thymus. In ...turn, thymocytes regulate the cellularity of Aire
mTEC
. However, it remains unknown whether thymocytes control the precursors of Aire
mTEC
that are contained in mTEC
cells or other mTEC
subsets that have recently been delineated by single-cell transcriptomic analyses. Here, using three distinct transgenic mouse models, in which antigen presentation between mTECs and CD4
thymocytes is perturbed, we show by high-throughput RNA-seq that self-reactive CD4
thymocytes induce key transcriptional regulators in mTEC
and control the composition of mTEC
subsets, including Aire
mTEC
precursors, post-Aire and tuft-like mTECs. Furthermore, these interactions upregulate the expression of tissue-restricted self-antigens, cytokines, chemokines, and adhesion molecules important for T-cell development. This gene activation program induced in mTEC
is combined with a global increase of the active H3K4me3 histone mark. Finally, we demonstrate that these self-reactive interactions between CD4
thymocytes and mTECs critically prevent multiorgan autoimmunity. Our genome-wide study thus reveals that self-reactive CD4
thymocytes control multiple unsuspected facets from immature stages of mTECs, which determines their heterogeneity.
Dugesia japonica planarian flatworms are naturally exposed to various microbes but typically survive this challenge. We show that planarians eliminate bacteria pathogenic to Homo sapiens, ...Caenorhabditis elegans, and/or Drosophila melanogaster and thus represent a model to identify innate resistance mechanisms. Whole-transcriptome analysis coupled with RNAi screening of worms infected with Staphylococcus aureus or Legionella pneumophila identified 18 resistance genes with nine human orthologs, of which we examined the function of MORN2. Human MORN2 facilitates phagocytosis-mediated restriction of Mycobacterium tuberculosis, L. pneumophila, and S. aureus in macrophages. MORN2 promotes the recruitment of LC3, an autophagy protein also involved in phagocytosis, to M. tuberculosis-containing phagosomes and subsequent maturation to degradative phagolysosomes. MORN2-driven trafficking of M. tuberculosis to single-membrane, LC3-positive compartments requires autophagy-related proteins Atg5 and Beclin-1, but not Ulk-1 and Atg13, highlighting the importance of MORN2 in LC3-associated phagocytosis. These findings underscore the value of studying planarian defenses to identify immune factors.
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•Dugesia japonica planarian flatworms eliminate a spectrum of ingested pathogenic bacteria•Transcriptomic and RNAi analyses reveal planarian genes promoting resistance to bacteria•MORN2 restricts the growth of all bacterial strains tested in planarians•MORN2 promotes lipidation of LC3-I and LC3-associated phagocytosis of M. tuberculosis
Dugesia japonica planarian flatworms are resistant to various bacteria that are pathogenic in humans. Abnave et al. use planarians as a model to identify antibacterial immune factors and determine that MORN2, conserved in Homo sapiens, restricts intracellular bacterial growth by promoting LC3-associated phagocytosis of invading bacteria.
Tropheryma whipplei, the agent of Whipple's disease, inhibits phago-lysosome biogenesis to create a suitable niche for its survival and replication in macrophages. To understand the mechanism by ...which it subverts phagosome maturation, we used biochemical and cell biological approaches to purify and characterise the intracellular compartment where Tropheryma whipplei resides using mouse bone-marrow-derived macrophages. We showed that in addition to Lamp-1, the Tropheryma whipplei phagosome is positive for Rab5 and Rab7, two GTPases required for the early to late phagosome transition. Unlike other pathogens, inhibition of PI(3)P production was not the mechanism for Rab5 stabilisation at the phagosome. Overexpression of the inactive, GDP-bound form of Rab5 bypassed the pathogen-induced blockade of phago-lysosome biogenesis. This suggests that Tropheryma whipplei blocks the switch from Rab5 to Rab7 by acting on the Rab5 GTPase cycle. A bio-informatic analysis of the Tropheryma whipplei genome revealed a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) homologous with the GAPDH of Listeria monocytogenes, and this may be the bacterial protein responsible for blocking Rab5 activity. To our knowledge, Tropheryma whipplei is the first pathogen described to induce a "chimeric" phagosome stably expressing both Rab5 and Rab7, suggesting a novel and specific mechanism for subverting phagosome maturation.
This protocol details the step-by-step procedure for in-depth immune phenotyping of peripheral blood natural killer (NK) cells from clinical samples by mass cytometry. The protocol consists of three ...main steps: PBMC incubation with a mix of metal-conjugated antibodies for extracellular phenotyping followed by fixation, permeabilization and incubation with a mix of metal-conjugated antibodies for staining of intracellular proteins, and sample acquisition on a mass cytometer. High-dimensional analysis enables the visualization of NK cell subsets and their phenotypical characteristics.
For complete details on the use and execution of this protocol, please refer to Chretien et al. (2021).
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•Protocol for deep phenotyping of human NK cells by mass cytometry•Sample staining with metal-conjugated antibodies•Sample acquisition on a mass cytometer•High-dimensional analysis using machine learning algorithms
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
This protocol details the step-by-step procedure for in-depth immune phenotyping of peripheral blood natural killer (NK) cells from clinical samples by mass cytometry. The protocol consists of three main steps: PBMC incubation with a mix of metal-conjugated antibodies for extracellular phenotyping followed by fixation, permeabilization and incubation with a mix of metal-conjugated antibodies for staining of intracellular proteins, and sample acquisition on a mass cytometer. High-dimensional analysis enables the visualization of NK cell subsets and their phenotypical characteristics.
The rationale for therapeutic targeting of Vδ2
γδ T cells in breast cancer is strongly supported by in vitro and murine preclinical investigations, characterizing them as potent breast tumor cell ...killers and source of Th1-related cytokines, backing cytotoxic αβ T cells. Nonetheless, insights regarding Vδ2
γδ T cell phenotypic alterations in human breast cancers are still lacking. This paucity of information is partly due to the challenging scarcity of these cells in surgical specimens. αβ T cell phenotypic alterations occurring in the tumor bed are detectable in the periphery and correlate with adverse clinical outcomes. Thus, we sought to determine through an exploratory study whether Vδ2
γδ T cells phenotypic changes can be detected within breast cancer patients' peripheral blood, along with association with tumor progression. By using mass cytometry, we quantified 130 immune variables from untreated breast cancer patients' peripheral blood. Supervised analyses and dimensionality reduction algorithms evidenced circulating Vδ2
γδ T cell phenotypic alterations already established at diagnosis. Foremost, terminally differentiated Vδ2
γδ T cells displaying phenotypes of exhausted senescent T cells associated with lymph node involvement. Thereby, our results support Vδ2
γδ T cells implication in breast cancer pathogenesis and progression, besides shedding light on liquid biopsies to monitor surrogate markers of tumor-infiltrating Vδ2
γδ T cell antitumor activity.
Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. ...Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.
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