Acral Melanoma Is Infiltrated with cDC1s and Functional Exhausted CD8 T Cells Similar to the Cutaneous Melanoma of Sun-Exposed Skin.

Acral melanoma (AM) is the most common melanoma in non-Caucasian populations, yet it remains largely understudied. As AM lacks the UV-radiation mutational signatures that characterize other cutaneous melanomas, it is considered devoid of immunogenicity and is rarely included in clinical trials assessing novel immunotherapeutic regimes aiming to recover the antitumor function of immune cells. We studied a Mexican cohort of melanoma patients from the Mexican Institute of Social Security (IMSS) (n = 38) and found an overrepresentation of AM (73.9%). We developed a multiparametric immunofluorescence technique coupled with a machine learning image analysis to evaluate the presence of conventional type 1 dendritic cells (cDC1) and CD8 T cells in the stroma of melanoma, two of the most relevant immune cell types for antitumor responses. We observed that both cell types infiltrate AM at similar and even higher levels than other cutaneous melanomas. Both melanoma types harbored programmed cell death protein 1 (PD-1+) CD8 T cells and PD-1 ligand (PD-L1+) cDC1s. Despite this, CD8 T cells appeared to preserve their effector function and expanding capacity as they expressed interferon-γ (IFN-γ) and KI-67. The density of cDC1s and CD8 T cells significantly decreased in advanced stage III and IV melanomas, supporting these cells' capacity to control tumor progression. These data also argue that AM could respond to anti-PD-1-PD-L1 immunotherapy.

Mast Cells in the Tumor Microenvironment.

Mast cells are tissue-resident, innate immune cells that play a key role in the inflammatory response and tissue homeostasis. Mast cells accumulate in the tumor stroma of different human cancer types, and increased mast cell density has been associated to either good or poor prognosis, depending on the tumor type and stage. Mast cells play a multifaceted role in the tumor microenvironment by modulating various events of tumor biology, such as cell proliferation and survival, angiogenesis, invasiveness, and metastasis. Moreover, tumor-associated mast cells have the potential to shape the tumor microenvironment by establishing crosstalk with other tumor-infiltrating cells. This chapter reviews the current understanding of the role of mast cells in the tumor microenvironment. These cells have received much less attention than other tumor-associated immune cells but are now recognized as critical components of the tumor microenvironment and could hold promise as a potential target to improve cancer immunotherapy.

An In Vitro Model of Mast Cell Recruitment and Activation by Breast Cancer Cells Supports Anti-Tumoral Responses.

Breast cancer (BrC) affects millions of women yearly. Mast cells (MCs) are common components of breast tumors with documented agonistic and antagonistic roles in tumor progression. Understanding the participation of MCs in BrC may lead to new therapies to control tumor growth. In this study, we looked into mechanistic models of MC responses triggered by BrC cells (BrCC), assessing both early degranulation and late transcriptional activities. We used aggressive and non-aggressive BrCC to model the progressive staging of the disease over HMC1 and LAD-2 human MC lines. We found that both MC lines were chemoattracted by all BrCC, but their activation was preferentially induced by aggressive lines, finding differences in their active transcriptional programs, both at basal level and after stimulation. Among those genes with altered expression were down-regulated SPP1, PDCD1, IL17A and TGFB1 and up-regulated KITLG and IFNG. A low expression of SPP1 and a high expression of KITLG and IFNG were associated with increased overall survival of BrC patients from public databases. The set of altered genes is more often associated with tumor stromas enriched with anti-tumoral signals, suggesting that MCs may participate in tumor control.

Giardia lamblia: identification of molecules that contribute to direct mast cell activation.

Mast cells play a central role in the early clearance of the intestinal parasite Giardia lamblia. In a previous study, we reported that G. lamblia live trophozoites or trophozoite-derived total soluble extract induced direct activation (IgE-independent) of mast cells and release of IL-6 and TNF-α. To identify the Giardia molecules and the mast cell receptors involved in this activation, trophozoite-derived total soluble proteins separated into three fractions (F1-F3) were evaluated for its ability to activate mast cells in vitro. F2 activated mast cells in a greater extent than F1 and F3. Furthermore, F2 induced the release of IL-6 and TNF-α by mast cells. TLR2 and TLR4 expression increased slightly after mast cell stimulation with either F2 or total soluble extract; however, these receptors were not involved in F2 or total soluble extract-induced proinflammatory cytokine production. Proteins present in F2 as unique and high-intensity bands identified by liquid chromatography coupled with tandem mass spectrometry, include molecules with important biological activities such as enolase and arginine deiminase (ADI). Recombinant ADI and enolase were tested for their ability to activate mast cells, but only ADI induced a significant release of IL-6 and TNF-α. ADI product, citrulline but not ammonium, also induced mast cell release of TNF-α. Interestingly, recombinant ADI still stimulated the secretion of TNF-α by mast cells in a arginine-free medium, although in a lower extend that in the presence of arginine, indicating that either ADI itself can stimulate mast cells or through its metabolic product, citrulline.

Structural analysis of the endogenous glycoallergen Hev b 2 (endo-ß-1,3-glucanase) from Hevea brasiliensis and its recognition by human basophils.

Endogenous glycosylated Hev b 2 (endo-ß-1,3-glucanase) from Hevea brasiliensis is an important latex allergen that is recognized by IgE antibodies from patients who suffer from latex allergy. The carbohydrate moieties of Hev b 2 constitute a potentially important IgE-binding epitope that could be responsible for its cross-reactivity. Here, the structure of the endogenous isoform II of Hev b 2 that exhibits three post-translational modifications, including an N-terminal pyroglutamate and two glycosylation sites at Asn27 and at Asn314, is reported from two crystal polymorphs. These modifications form a patch on the surface of the molecule that is proposed to be one of the binding sites for IgE. A structure is also proposed for the most important N-glycan present in this protein as determined by digestion with specific enzymes. To analyze the role of the carbohydrate moieties in IgE antibody binding and in human basophil activation, the glycoallergen was enzymatically deglycosylated and evaluated. Time-lapse automated video microscopy of basophils stimulated with glycosylated Hev b 2 revealed basophil activation and degranulation. Immunological studies suggested that carbohydrates on Hev b 2 represent an allergenic IgE epitope. In addition, a dimer was found in each asymmetric unit that may reflect a regulatory mechanism of this plant defence protein.

The cholera toxin B subunit induces trained immunity in dendritic cells and promotes CD8 T cell antitumor immunity.

Introduction: Innate immune training is a metabolic, functional, and epigenetic long-term reprogramming of innate cells triggered by different stimuli. This imprinting also reaches hematopoietic precursors in the bone marrow to sustain a memory-like phenotype. Dendritic cells (DCs) can exhibit memory-like responses, enhanced upon subsequent exposure to a pathogen; however, whether this imprinting is lineage and stimulus-restricted is still being determined. Nevertheless, the functional consequences of DCs training on the adaptive and protective immune response against non-infectious diseases remain unresolved. Methods: We evaluated the effect of the nontoxic cholera B subunit (CTB), LPS and LTA in the induction of trained immunity in murine DCs revealed by TNFa and LDH expression, through confocal microscopy. Additionally, we obtained bone marrow DCs (BMDCs) from mice treated with CTB, LPS, and LTA and evaluated training features in DCs and their antigen-presenting cell capability using multiparametric cytometry. Finally, we design an experimental melanoma mouse model to demonstrate protection induced by CTB-trained DCs in vivo. Results: CTB-trained DCs exhibit increased expression of TNFa, and metabolic reprogramming indicated by LDH expression. Moreover, CTB training has an imprint on DC precursors, increasing the number and antigen-presenting function in BMDCs. We found that training by CTB stimulates the recruitment of DC precursors and DCs infiltration at the skin and lymph nodes. Interestingly, training-induced by CTB promotes a highly co-stimulatory phenotype in tumor-infiltrating DCs (CD86+) and a heightened functionality of exhausted CD8 T cells (Ki67+, GZMB+), which were associated with a protective response against melanoma challenge in vivo. Conclusion: Our work indicates that CTB can induce innate immune training on DCs, which turns into an efficient adaptive immune response in the melanoma model and might be a potential immunotherapeutic approach for tumor growth control.

Mast-Cell Response to Leishmania mexicana and Sand-Fly Salivary Proteins Is Modulated by Orchiectomy.

Mast cells (MCs) play a crucial role during Leishmania infections, which is transmitted through the bite of an infected sand fly that injects saliva together with the parasite. Sand fly saliva is a complex fluid that modulates the host immune response. In addition, hormonal factors modulate the host immune response and alter susceptibility to infections. Thus, to assess the impact of male sex hormones on the mast-cell (MC) response to Leishmania infections, we orchiectomized male mice, infected them with the parasite in the presence of sand fly salivary proteins, and analyzed the inflammatory response of MCs. Our results showed that the MC response to the parasite and vector salivary proteins differed between orchiectomized and sham-operated mice. In orchiectomized mice, MC showed a retarded activation pattern, associated with slower degranulation and weaker TNF-α, histamine, and tryptase staining in response to the infection with Leishmania mexicana combined with vector-salivary proteins, as compared to sham mice. Furthermore, neutrophil infiltration was slower in orchiectomized mice, and numbers of infected macrophages and lesion sizes were smaller. Our results show that, during Leishmania infection, male sex hormones modulate the mast-cell response against the parasite and salivary proteins of the sand fly vector, inducing an intense inflammatory response. Their absence in orchiectomized mice retards the inflammatory response, enabling better control of the infection and slower disease progression.

Giardia lamblia: interleukin 6 and tumor necrosis factor-alpha release from mast cells induced through an Ig-independent pathway.

Giardia lamblia is a common cause of both acute and chronic diarrheal disease in humans worldwide. It has been shown that mast cells, IL-6 and TNF-alpha are substantially involved in the early control of G. lamblia infection in mice. However, no studies have yet been reported concerning the interaction between mast cell and Giardia, as well as the mast cells mediators generated in response to Giardia infection. In this study we demonstrated the direct activation of mast cells by G. lamblia live trophozoites or trophozoite-derived antigens followed by an increase in tryptase expression and a significant release of the preformed mediator histamine. In addition, parasite derived antigens increased TNF-alpha and de novo synthesized cytokine IL-6, at the mRNA and protein level. These results strongly suggest that mast cells might be an important source not only of IL-6 but also of TNF-alpha during Giardia infection, playing an important role in the outcome of the infection.

Potential Novel Risk Factor for Breast Cancer: Toxocara canis Infection Increases Tumor Size Due to Modulation of the Tumor Immune Microenvironment.

Worldwide, breast cancer is the most important type of cancer in women with regard to incidence and prevalence. Several risk factors interact to increase the probability of breast cancer development. Biological environmental contaminants such as infectious agents play a significant role in tumor development, and helminths have been recognized as cancer enhancers or inducers due to their ability to regulate the host immune response. Toxocara canis is a zoonotic and cosmopolite nematode with immuno-regulatory abilities. T. canis infection has been related to T helper type-2 cell (Th2 or type 2) and regulatory responses. Type 2 and regulatory immune responses may favor the development of comorbidities that are usually controlled or eliminated through a type 1 response such as cancer. The aim of this study was to determine whether T. canis infection alters mammary tumor growth through modulation of the immune response. Infected mice developed larger tumors. Tumor immune cell milieu analysis revealed that infection reduced the proportions of CD8+ lymphocytes and increased the proportions of F4/80+ macrophages and CD19+ B cells. These changes were accompanied by a type 2 local response represented by increased amounts of IL-4 and VEGF and a regulatory microenvironment associated with higher IL-10 levels. Thus, this study demonstrates that T. canis infection enhances tumor development and suggests that this is through modulation of the tumor immune microenvironment.

The histone deacetylase inhibitor valproic acid attenuates phospholipase Cγ2 and IgE-mediated mast cell activation.

Mast cell activation through the high-affinity IgE receptor (FcεRI) plays a central role in allergic reactions. FcεRI-mediated activation triggers multiple signaling pathways leading to degranulation and synthesis of different inflammatory mediators. IgE-mediated mast cell activation can be modulated by different molecules, including several drugs. Herein, we investigated the immunomodulatory activity of the histone deacetylase inhibitor valproic acid (VPA) on IgE-mediated mast cell activation. To this end, bone marrow-derived mast cells (BMMC) were sensitized with IgE and treated with VPA followed by FcεRI cross-linking. The results indicated that VPA reduced mast cell IgE-dependent degranulation and cytokine release. VPA also induced a significant reduction in the cell surface expression of FcεRI and CD117, but not other mast cell surface molecules. Interestingly, VPA treatment inhibited the phosphorylation of PLCγ2, a key signaling molecule involved in IgE-mediated degranulation and cytokine secretion. However, VPA did not affect the phosphorylation of other key components of the FcεRI signaling pathway, such as Syk, Akt, ERK1/2, or p38. Altogether, our data demonstrate that VPA affects PLCγ2 phosphorylation, which in turn decreases IgE-mediated mast cell activation. These results suggest that VPA might be a key modulator of allergic reactions and might be a promising therapeutic candidate.

Mast Cell, the Neglected Member of the Tumor Microenvironment: Role in Breast Cancer.

Mast cells are unique tissue-resident immune cells that secrete a diverse array of biologically active compounds that can stimulate, modulate, or suppress the immune response. Although mounting evidence supports that mast cells are consistently infiltrating tumors, their role as either a driving or an opposite force for cancer progression is still controversial. Particularly, in breast cancer, their function is still under discussion. While some studies have shown a protective role, recent evidence indicates that mast cells enhance blood and lymphatic vessel formation. Interestingly, one of the most important components of the mast cell cargo, the serine protease tryptase, is a potent angiogenic factor, and elevated serum tryptase levels correlate with bad prognosis in breast cancer patients. Likewise, histamine is known to induce tumor cell proliferation and tumor growth. In agreement, mast cell depletion reduces the size of mammary tumors and metastasis in murine models that spontaneously develop breast cancer. In this review, we will discuss the evidence supporting protumoral and antitumoral roles of mast cells, emphasizing recent findings placing mast cells as important drivers of tumor progression, as well as the potential use of these cells or their mediators as therapeutic targets.

Gender-related effects of sex steroids on histamine release and FcεRI expression in rat peritoneal mast cells.

Mast cells (MCs) are versatile effector and regulatory cells in various physiologic, immunologic, and pathologic processes. In addition to the well-characterized IgE/FcεRI-mediated degranulation, a variety of biological substances can induce MCs activation and release of their granule content. Sex steroids, mainly estradiol and progesterone, have been demonstrated to elicit MCs activation. Most published studies have been conducted on MCs lines or freshly isolated peritoneal and bone marrow-derived MC without addressing gender impact on MC response. Our goal was to investigate if the effect of estradiol, progesterone, testosterone, and dihydrotestosterone (DHT) on MCs may differ depending on whether female or male rats are used as MCs donors. Our results demonstrated that effect of sex steroids on MCs histamine release is dose- and gender-dependent and can be direct, synergistic, or inhibitory depending on whether hormones are used alone or to pretreat MCs followed by substance P-stimulation or upon IgE-mediated stimulation. In contrast, sex steroids did not have effect on the MC expression of the IgE high affinity receptor, FcεRI, no matter female or male rats were used. In conclusion, MCs degranulation is modulated by sex hormones in a gender-selective fashion, with MC from females being more susceptible than MC from males to the effects of sex steroids.

Recognition of Candida albicans by Dectin-1 induces mast cell activation.

Mast cells are crucial elements of the innate immune response. They reside in tissues that are commonly exposed to the external environment, such as the skin and mucosae, where they can rapidly detect the presence of pathogens and mount a potent inflammatory response that recruits other cellular effectors of the immune response. The contribution of mast cells to the immune response to viruses, bacteria, protozoa and multicellular parasites is well established, but there is scarce information about the role of these cells in fungal infections. In this study, we analyzed if mast cells are activated by Candida albicans and if the C-type lectin receptor Dectin-1 is involved in its recognition. We found that both yeasts and hyphae of C. albicans-induced mast cell degranulation and production of TNF-α, IL-6, IL-10, CCL3 and CCL4, while only yeasts were able to induce IL-1ß. Mast cells also produced ROS after stimulation with both dimorphic phases of C. albicans. When mast cells were activated with yeasts and hyphae, they showed decreased expression of IκBα and increased presence of phosphorylated Syk. Blockade of the receptor Dectin-1, but not Toll-like receptor 2, decreased TNF-α production by mast cell in response to C. albicans. These results indicate that mast cells are capable of sensing the two phases of C. albicans, and suggest that mast cells participate as an early inductor of inflammation during the early innate immune response to this fungus.

Non-reproductive effects of sex steroids: their immunoregulatory role.

Sex hormones play an important role as modulators of the immune system. A growing body of evidence shows reciprocal relationship between sex steroids and the immune system. Since the innate immune response determines the type of adaptive immune response, hormonal effects on the innate immune response may affect subsequent adaptive immunity. The sex steroids estrogens, progesterone and testosterone regulate growth, differentiation, survival and function of many cell types involved in homeostasis and immunity. The presence of sex steroid receptors on immune cells indicates that sex steroids may exert their biological effects by binding to these receptors. Sex steroids and immunity are closely connected, and their mutual regulation is involved in the maintenance of immune balance. Understanding the mechanisms of action of sex steroids on immune cells is important for further progress in the development of novel therapies for chronic diseases associated to immune dysregulation. This review describes the effects of sex steroids on the different immune system cells, and the possible implications of these effects on the incidence of many diseases.