New Insight into Intestinal Mast Cells Revealed by Single-Cell RNA Sequencing

Mast cells (MCs) are tissue-resident immune cells distributed in all tissues and strategically located close to blood and lymphatic vessels and nerves. Thanks to the expression of a wide array of receptors, MCs act as tissue sentinels, able to detect the presence of bacteria and parasites and to respond to different environmental stimuli. MCs originate from bone marrow (BM) progenitors that enter the circulation and mature in peripheral organs under the influence of microenvironment factors, thus differentiating into heterogeneous tissue-specific subsets. Even though MC activation has been traditionally linked to IgE-mediated allergic reactions, a role for these cells in other pathological conditions including tumor progression has recently emerged. However, several aspects of MC biology remain to be clarified. The advent of single-cell RNA sequencing platforms has provided the opportunity to understand MCs’ origin and differentiation as well as their phenotype and functions within different tissues, including the gut. This review recapitulates how single-cell transcriptomic studies provided insight into MC development as well as into the functional role of intestinal MC subsets in health and disease

NKG2D down-modulation on human NK cells is followed by DNAM-1 hypo-responsivenss

Natural killer (NK) cells are cytotoxic innate lymphocytes that represent the first line of defense against viral infections and tumor growth. NK cell activation is regulated by activating receptors able to recognize self-molecules up-regulated in stress conditions and inhibitory receptors that mainly bind to major histocompatibility complex class-I (MHC-I) molecules to prevent lysis of normal cells. Among activating receptors, NKG2D and DNAM-1 play a pivotal role in anticancer immune responses since they bind ligands upregulated on transformed cells. The interaction of both NKG2D and DNAM-1 with their ligands promotes a rapid receptor downmodulation that mainly occurs through internalization and lysosomal degradation. Particularly, NKG2D downmodulation has been associated to an exhausted phenotype characterized by down-modulation of the cytolytic machinery and upregulation of inhibitory receptors on both human and murine NK cells. However, our knowledge of the consequences of NKG2D engagement is still incomplete. The aim of the study is to investigate if NKG2D downmodulation could impair DNAM-1 functionality. Primary human NK cells were co-cultured with MICA-transfectants, purified, and used as effector cells in a cytotoxicity assay toward Ba/F3 cell line stably overexpressing MICA and PVR. MICA-experienced NK cells resulted impaired in NKG2D-mediated killing and showed a marked reduction of DNAM-1-triggered cytotoxicity. Analysing the different stages of cellular cytotoxicity by confocal microscopy, we found that MICA-experienced NK cells retained the ability to induce LFA-1 conformational change upon PVR recognition while they fail to polarize perforin-containing granules toward the BaF/3-PVR contact site. We verified the expression of DNAM-1-related inhibitory receptors, CD96 and TIGIT, and found that while CD96 expression remains untouched, TIGIT resulted upregulated in MICA-experienced NK cells both at protein and mRNA levels, as demonstrated by employing FACS and Real-Time PCR analyses. We performed cytotoxicity assays and experiments of confocal microscopy in the presence of anti-TIGIT blocking antibody demonstrating TIGIT contribution in inhibiting the killing of Ba/F3-PVR and a in perforin polarization impairment. However, impairment of MICA-experienced NK cell ability to lyse PVR-expressing target cells was only partially reverted by TIGIT blocking Ab, suggesting that besides TIGIT other mechanisms are responsible for defective lysis. Thus, MICA-experienced NK cells were cross-linked with anti-DNAM-1 antibody and signaling events leading to NK cell killing were assessed. Immunoblotting of cell lysates showed that, Vav1 and Akt phosphorylation resulted almost unaltered. However, DNAM-1 mediated activation of Erk1/2 and Pyk2 was reduced demonstrating that NKG2D stimulation directly affects some of the DNAM-1 activating signals leading to cell cytotoxicity. Collectively, these results demonstrate that NKG2D engagement on human NK cells impairs DNAM-1-mediated killing through two different converging mechanisms: by the upregulation of the checkpoint inhibitory receptor TIGIT, that in turn suppresses DNAM-1-mediated cytotoxic function, and by direct inhibition of DNAM-1-promoted signaling. Our results highlight a novel interplay between NKG2D and DNAM-1/TIGIT receptors that may facilitate neoplastic cell evasion from NK cell-mediated clearance

SCF and IL-33 regulate mouse mast cell phenotypic and functional plasticity supporting a pro-inflammatory microenvironment

Abstract Mast cells (MCs) are multifaceted innate immune cells often present in the tumor microenvironment (TME). Several recent findings support their contribution to the transition from chronic inflammation to cancer. However, MC-derived mediators can either favor tumor progression, inducing the spread of the tumor, or exert anti-tumorigenic functions, limiting tumor growth. This apparent controversial role likely depends on the plastic nature of MCs that under different microenvironmental stimuli can rapidly change their phenotype and functions. Thus, the exact effect of unique MC subset(s) during tumor progression is far from being understood. Using a murine model of colitis-associated colorectal cancer, we initially characterized the MC population within the TME and in non-lesional colonic areas, by multicolor flow cytometry and confocal microscopy. Our results demonstrated that tumor-associated MCs harbor a main connective tissue phenotype and release high amounts of Interleukin (IL)-6 and Tumor Necrosis Factor (TNF)-α. This MC phenotype correlates with the presence of high levels of Stem Cell Factor (SCF) and IL-33 inside the tumor. Thus, we investigated the effect of SCF and IL-33 on primary MC cultures and underscored their ability to shape MC phenotype eliciting the production of pro-inflammatory cytokines. Our findings support the conclusion that during colonic transformation a sustained stimulation by SCF and IL-33 promotes the accumulation of a prevalent connective tissue-like MC subset that through the secretion of IL-6 and TNF-α maintains a pro-inflammatory microenvironment

Artificial Intelligence in Dermatopathology: New Insights and Perspectives

In recent years, an increasing enthusiasm has been observed towards artificial intelligence and machine learning, involving different areas of medicine. Among these, although still in the embryonic stage, the dermatopathological field has also been partially involved, with the attempt to develop and train algorithms that could assist the pathologist in the differential diagnosis of complex melanocytic lesions. In this article, we face this new challenge of the modern era, carry out a review of the literature regarding the state of the art and try to determine promising future perspectives