Atherosclerosis, a common age-related disease, is characterized by intense immunological activity. Atherosclerotic plaque is composed of endothelial cells, vascular smooth muscle cells (VSMCs), ...lipids and immune cells infiltrating from the blood. During progression of the disease, VSMCs undergo senescence within the plaque and secrete SASP (senescence-associated secretory phenotype) factors that can actively modulate plaque microenvironment. We demonstrated that senescent VSMCs secrete increased number of extracellular vesicles (senEVs). Based on unbiased proteomic analysis of VMSC-derived EVs and of the soluble fraction of SASP (sSASP), more than 900 proteins were identified in each of SASP compartments. Comparison of the composition of VMSC-derived EVs with the SASP atlas revealed several proteins, including Serpin Family F Member 1 (SERPINF1) and Thrombospondin 1 (THBS1), as commonly upregulated components of EVs secreted by senescent VSMCs and fibroblasts. Among soluble SASP factors, only Growth Differentiation Factor 15 (GDF15) was universally increased in the secretome of senescent VSMCs, fibroblasts, and epithelial cells. Bioinformatics analysis of EV proteins distinguished functionally organized protein networks involved in immune cell function regulation. Accordingly, EVs released by senescent VSMCs induced secretion of IL-17, INFγ, and IL-10 by T cells and of TNFα produced by monocytes. Moreover senEVs influenced differentiation of monocytes favoring mix M1/M2 polarization with proinflammatory characteristics. Altogether, our studies provide a complex, unbiased analysis of VSMC SASP and prove that EVs derived from senescent VSMCs influence the cytokine milieu by modulating immune cell activity. Our results strengthen the role of senescent cells as an important inducer of inflammation in atherosclerosis.
Previously, we showed that mouse delayed-type hypersensitivity (DTH) can be antigen-specifically downregulated by suppressor T cell-derived miRNA-150 carried by extracellular vesicles (EVs) that ...target antigen-presenting macrophages. However, the exact mechanism of the suppressive action of miRNA-150-targeted macrophages on effector T cells remained unclear, and our current studies aimed to investigate it. By employing the DTH mouse model, we showed that effector T cells were inhibited by macrophage-released EVs in a miRNA-150-dependent manner. This effect was enhanced by the pre-incubation of EVs with antigen-specific antibodies. Their specific binding to MHC class II-expressing EVs was proved in flow cytometry and ELISA-based experiments. Furthermore, by the use of nanoparticle tracking analysis and transmission electron microscopy, we found that the incubation of macrophage-released EVs with antigen-specific antibodies resulted in EVs’ aggregation, which significantly enhanced their suppressive activity in vivo. Nowadays, it is increasingly evident that EVs play an exceptional role in intercellular communication and selective cargo transfer, and thus are considered promising candidates for therapeutic usage. However, EVs appear to be less effective than their parental cells. In this context, our current studies provide evidence that antigen-specific antibodies can be easily used for increasing EVs’ biological activity, which has great therapeutic potential.
Cancer continues to be the leading cause of mortality in high-income countries, necessitating the development of more precise and effective treatment modalities. Immunotherapy, specifically adoptive ...cell transfer of T cell receptor (TCR)-engineered T cells (TCR-T therapy), has shown promise in engaging the immune system for cancer treatment. One of the biggest challenges in the development of TCR-T therapies is the proper prediction of the pairing between TCRs and peptide-human leukocyte antigen (pHLAs). Modern computational immunology, using artificial intelligence (AI)-based platforms, provides the means to optimize the speed and accuracy of TCR screening and discovery.
This study proposes an observational clinical trial protocol to collect patient samples and generate a database of pHLA:TCR sequences to aid the development of an AI-based platform for efficient selection of specific TCRs.
The multicenter observational study, involving 8 participating hospitals, aims to enroll patients diagnosed with stage II, III, or IV colorectal cancer adenocarcinoma.
Patient recruitment has recently been completed, with 100 participants enrolled. Primary tumor tissue and peripheral blood samples have been obtained, and peripheral blood mononuclear cells have been isolated and cryopreserved. Nucleic acid extraction (DNA and RNA) has been performed in 86 cases. Additionally, 57 samples underwent whole exome sequencing to determine the presence of somatic mutations and RNA sequencing for gene expression profiling.
The results of this study may have a significant impact on the treatment of patients with colorectal cancer. The comprehensive database of pHLA:TCR sequences generated through this observational clinical trial will facilitate the development of the AI-based platform for TCR selection. The results obtained thus far demonstrate successful patient recruitment and sample collection, laying the foundation for further analysis and the development of an innovative tool to expedite and enhance TCR selection for precision cancer treatments.
ClinicalTrials.gov NCT04994093; https://clinicaltrials.gov/ct2/show/NCT04994093.
DERR1-10.2196/45872.
Tumour-derived microvesicles (TMVs) may interact with cells of the immune system. Our previous observations indicated that TMVs modulate production of cytokines and reactive oxygen species (ROS) by ...monocytes. This study was designed to determine the role of TMVs in stimulation of chemokine production by human monocytes.
Chemokines at the mRNA and protein level were detected by real-time PCR and by Western blot, respecively. Chemokine release and chemotaxis of blood leukocytes were analysed by flow cytometry. Matrigel assay was used to determine angiogenesis in a NOD-SCID mice model.
TMVs induced secretion of interleukin-8 (CXCL8), monocyte chemoattractant protein-1 (CCL2), macrophage inflammatory protein-1α (CCL3) and MIP-1β (CCL4), and regulated on activation normal T-cells expressed and secreted (CCL5) chemokines and accumulation of their mRNA in monocytes. Moreover, TMVs enhanced angiogenesis in NOD-SCID mice by delivering chemokines and via stimulation of monocytes. In addition, TMVs may be storage for chemokines thus inducing chemotaxis of blood leukocytes.
These results further support the role of TMVs in modulation of monocyte biological activity.
The tumour microenvironment represents a dynamic complex milieu, which includes tumour cells, cells of the immune system and other (cellular and non-cellular) components. The role of these particular ...'puzzle pieces' may change substantially due to their mutual interactions. The present review concerns different opinions on interactions that occur between monocytes, tumour cells and TMVs (tumour-derived microvesicles).
Monocytes exhibit direct and indirect antitumour activities and may be potentially useful for various forms of adoptive cellular immunotherapy of cancer. However, blood is a limited source of them. ...This study explored whether monocytes can be obtained from bone marrow haematopoietic CD34
+
stem cells of colon cancer patients, using previously described protocol of expansion and differentiation to monocytes of cord blood-derived CD34
+
haematopoietic progenitors. Data show that in two-step cultures, the yield of cells was increased approximately 200-fold, and among these cells, up to 60 % of CD14
+
monocytes were found. They consisted of two subpopulations: CD14
++
CD16
+
and CD14
+
CD16
−
, at approximately 1:1 ratio, that differed in HLA-DR expression, being higher on the former. No differences in expression of costimulatory molecules were observed, as CD80 was not detected, while CD86 expression was comparable. These CD14
+
monocytes showed the ability to present recall antigens (PPD,
Candida albicans
) and neoantigens expressed on tumour cells and tumour-derived microvesicles (TMV) to autologous CD3
+
T cells isolated from the peripheral blood. Monocytes also efficiently presented the immunodominant HER-2/neu
369–377
peptide (KIFGSLAFL), resulting in the generation of specific cytotoxic CD8
+
T lymphocytes (CTL). The CD14
++
CD16
+
subset exhibited enhanced cytotoxicity, though nonsignificant, towards tumour cells in vitro. These observations indicate that generation of monocytes from CD34
+
stem cells of cancer patients is feasible. To our knowledge, it is the first demonstration of such approach that may open a way to obtain autologous monocytes for alternative forms of adaptive and adoptive cellular immunotherapy of cancer.
Tumour-derived microvesicles (TMVs) are important players in tumour progression, modulating biological activity of immune cells e.g. lymphocytes, monocytes and macrophages. This phenomenon is ...particularly interesting in the progression of colon cancer, as macrophages in this type of tumour are relevant for the recovery processes. In the present study, the role of colon cancer cell-derived microvesicles in monocyte differentiation and activity profile (polarization) was investigated.
Monocyte-derived macrophages (MDM) were differentiated in vitro in the presence of TMVs obtained from colon cancer: Caco-2, SW620, LoVo or SW480 cell lines and analysed according to their morphology and biological functions, as defined by cytokine secretion, reactive oxygen intermediate (ROI) production and cytotoxic activity against respective colon cancer cells.
Monocytes differentiated with TMVs exhibited morphological and phenotypical characteristics of macrophages. An early contact (beginning with the first day of the in vitro culture) of monocytes with TMVs resulted in increased IL-10 secretion and only slightly elevated TNF release. Early, or prolonged contact resulted in low ROI production and low cytotoxicity against tumour cells. On the other hand, late contact of MDM with TMVs, stimulated MDM to significant TNF and IL-12 secretion, ROI production and enhanced cytotoxicity against tumour cells in vitro. In addition, differences in MDM response to TMVs from different cell lines were observed (according to cytokine secretion, ROI production and cytotoxicity against tumour cells in vitro). Biological activity, STATs phosphorylation and microRNA profiling of MDMs indicated differences in their polarization/activation status which may suggest mixed polarization type M1/M2 with the predominance of proinflammatory cells after late contact with TMVs.
Macrophage activity (polarization status) may be regulated by contact with not only tumour cells but also with TMVs. Their final polarization status depends on the contact time, and probably on the vesicle "cargo", as signified by the distinct impact of TMVs which enabled the switching of MDM maturation to regulatory macrophages.
Although blood monocytes exhibit significant cytotoxic activity against tumor cells, the function of tumor infiltrating macrophages (TIM) is depressed in cancer patients. This study addresses the ...question of how the antitumor response of human monocytes, assessed by production of cytokines (tumor necrosis factor alpha, TNF; IL-10; IL-12p40) and cytotoxicity, is altered by exposure to cancer cells. Tumor cell--pre-exposed monocytes restimulated with tumor cells showed significantly decreased production of TNF, IL-12, increased IL-10 (mRNA and release) and inhibition of IL-1 receptor-associated kinase-1 (IRAK-1) expression. This down-regulation of cytokine production was selective, as the response of pre-exposed monocytes to lipopolysaccharide (LPS) was unaffected. Treatment of tumor cell--pre-exposed monocytes with hyaluronidase (HAase) improved their depressed production of TNF, while HAase-treated cancer cells did not cause monocyte dysfunction. The response of hyaluronan (HA)--pre-exposed monocytes to stimulation with tumor cells was also inhibited. Cytotoxic activity of monocytes pretreated with cancer cells was also decreased. This study shows that tumor cells selectively deactivate monocytes and suggests that tumor cell-derived HA by blocking CD44 on monocytes inhibits their antitumor response. These observations may provide some explanation for the depressed function of TIM in human malignancy.
This study was designed to determine the characteristics of tumour cell-derived microvesicles (TMV) and their interactions with human monocytes. TMV were shed spontaneously by three different human ...cancer cell lines but their release was significantly increased upon activation of the cells with phorbol 12-myristate 13-acetate (PMA). TMV showed the presence of several surface determinants of tumour cells, e.g. HLA class I, CD29, CD44v7/8, CD51, chemokine receptors (CCR6, CX3CR1), extracellular matrix metalloproteinase inducer (EMMPRIN), epithelial cell adhesion molecule (EpCAM), but their level of expression differed from that on cells they originated from. TMV also carried mRNA for growth factors: vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), interleukin-8 (IL-8) and surface determinants (CD44H). TMV were localized at the monocytes surface following their short exposure to TMV, while at later times intracellularly. TMV transferred CCR6 and CD44v7/8 to monocytes, exerted antiapoptotic effect on monocytes and activated AKT kinase (Protein Kinase B). Thus, TMV interact with monocytes, alter their immunophenotype and biological activity. This implicates the novel mechanism by which tumour infiltrating macrophages may be affected by tumour cells not only by a direct cell to cell contact, soluble factors but also by TMV.
Abstract Tumour cells are shedding membrane fragments (tumour-derived microvesicles, TMV) that may interact with cells of immune system. Our previous observations indicated that TMV carry several ...surface determinants and mRNA of tumour cells and transfer some of them to monocytes. This study determined the effect of TMV on biological activity of human monocytes as the precursors of tumour infiltrating macrophages (TIM). It was found that TMV activated monocytes as shown by an increased HLA-DR expression, induced production of ROI (reactive oxygen intermediates) and of tumour necrosis factor (TNF), interleukin (IL)-10, IL-12p40 accumulation of mRNA and their secretion. Induction of TNF synthesis was CD44 dependent as blocking of CD44 on monocytes abolished its secretion. TMV-treated monocytes showed an increased antitumour activity as judged by enhanced cytotoxicity/cytostasis against tumour cells in vitro . Taken together, these results indicate that TMV significantly modulate biological activity of monocytes and thus mimic the effect of tumour cells on them. This may suggest that tumour cells interact with TIM not only via direct contact, soluble factors, but also TMV.
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