Thrombomodulin (TM) is a promising natural anti-coagulant therapeutic protein that is effective in the treatment of disseminated intravascular coagulation. However, the mechanisms by which TM on micro-vessels enable the regulation of intimal hyperplasia remain elusive. We investigated the graft-protective effects of TM in a fully major histocompatibility complex-mismatched murine cardiac allograft transplantation model. CBA recipients transplanted with a C57BL/6 heart received intraperitoneal administration of 0.2, 2.0, and 20.0 μg/day of TM for 8 days. Histological staining was conducted to assess the degree of inflammation and infiltration in the transplanted cardiac grafts. Untreated CBA recipients rejected C57BL/6 cardiac grafts acutely (median survival time [MST] was 7 days). CBA recipients exposed to the above dosages had significantly prolonged allograft survival (MSTs were 16, 21, and 37.5 days, respectively). Histologic assessments from TM-exposed recipients 2 weeks after grafting showed that the myocardium and vessel structure in their allografts were clearly preserved, and that the infiltration of inflammatory cells around coronary arteries was suppressed. TM can induce the prolongation of fully major histocompatibility complex-mismatched cardiac allograft by exerting graft protective effects within the myocardium and coronary arteries.

Background: Plasmodium falciparum malaria increases plasma levels of the cytokine Fms-like tyrosine kinase 3 ligand (Flt3L), a haematopoietic factor associated with dendritic cell (DC) expansion. It is unknown if the zoonotic parasite Plasmodium knowlesi impacts Flt3L or DC in human malaria. This study investigated circulating DC and Flt3L associations in adult malaria and in submicroscopic experimental infection.

Methods: Plasma Flt3L concentration and blood CD141⁺ DC, CD1c⁺ DC and plasmacytoid DC (pDC) numbers were assessed in (i) volunteers experimentally infected with P. falciparum and in Malaysian patients with uncomplicated (ii) P. falciparum or (iii) P. knowlesi malaria.

Results: Plasmodium knowlesi caused a decline in all circulating DC subsets in adults with malaria. Plasma Flt3L was elevated in acute P. falciparum and P. knowlesi malaria with no increase in a subclinical experimental infection. Circulating CD141⁺ DCs, CD1c⁺ DCs and pDCs declined in all adults tested, for the first time extending the finding of DC subset decline in acute malaria to the zoonotic parasite P. knowlesi.

Conclusions: In adults, submicroscopic Plasmodium infection causes no change in plasma Flt3L but does reduce circulating DCs. Plasma Flt3L concentrations increase in acute malaria, yet this increase is insufficient to restore or expand circulating CD141⁺ DCs, CD1c⁺ DCs or pDCs. These data imply that haematopoietic factors, yet to be identified and not Flt3L, involved in the sensing/maintenance of circulating DC are impacted by malaria and a submicroscopic infection. The zoonotic P. knowlesi is similar to other Plasmodium spp in compromising DC in adult malaria.

Keywords: BDCA1; BDCA3; CD141; CHMI; Dendritic cells; Flt3 ligand; IBSM; Plasmacytoid; Plasmodium falciparum; Plasmodium knowlesi.

Dendritic cells (DC) are cells of hematopoietic origin, which constitutively express MHC class I and II, and are functionally the most potent inducers of T-lymphocyte activation and proliferation. CD8+ T lymphocytes proliferate and acquire cytotoxic functions upon recognition of their cognate antigen on the surface of one or various dendritic cells with which they interact. However, only some DC subsets are able to present antigen to cytotoxic T cell precursors as taken up from extracellular sources. This function is termed cross-presentation (in Spanish, presentación cruzada or presentación subrogada) and requires shuttle mechanisms from phagosomes to the cytosol for antigen processing. It has been demonstrated that the differentiation of DC with these capabilities is dependent on FLT-3L and the transcription factor BATF3. They express peculiar functions and differentiation markers. These cells are distinguished in mice by surface CD8 features, while CD141 (BDCA-3) marks these cells in the human. These subpopulations are capable of selective internalization of necrotic cell debris by means of their CLEC9A lectin which is a receptor for extracellular polymerized actin. Expression of the chemokine receptor XCR1 favours contact with CD8+ T cells. Therapeutic vaccination with tumour antigens using DC is a strategy under development for the treatment of cancer. The use of DC subsets with more prominent capabilities for cross-presentation would mimic the natural mechanisms of immunization to induce cytolitic T lymphocytes. In vivo targeting of antigens with monoclonal antibodies against DEC-205 or CLEC9A attains very robust immune responses and is a strategy undergoing clinical trials for chronic viral diseases and malignancies.

Classic dendritic cells (cDCs) play a central role in the immune system by processing and presenting antigens to activate T cells, and consist of two major subsets: CD141⁺ cDC (cDC1) and CD1c⁺ cDC (cDC2). A population of migratory precursor cells, the pre-cDCs, is the immediate precursors to both cDC subsets. Previous studies showed that there were two pre-committed pre-cDC subpopulations. However, the key molecular drivers of pre-commitment in human pre-cDCs were not investigated.

To identify the key molecular drivers for pre-commitment in human pre-cDCs, we performed single cell RNA sequencing (RNA-Seq) of two cDC subsets and pre-cDCs, and bulk RNA-Seq of pre-cDCs and cDCs from human peripheral blood. We found that pre-DC subpopulations cannot be separated by either variable genes within pre-cDCs or differentially expressed genes between cDC1 and cDC2. In contrast, they were separated by 16 transcription factors that are themselves differentially expressed or have regulated targets enriched in the differentially expressed genes between bulk cDC1 and cDC2, with one subpopulation close to cDC1 and the other close to cDC2. More importantly, these two pre-cDC sub-populations are correlated with ratio of IRF8 to IRF4 expression level more than their individual expression level. We also verified these findings using three recently published datasets.In this study, we demonstrate that single cell transcriptome profiling can reveal pre-cDCs differentiation map, and our results suggest the concept that combinatorial dose of transcription factors determines cell differentiation fate.

Raynaud's phenomenon (RP) is the earliest and most common clinical manifestation in patients with systemic sclerosis (SSc) and its related diseases containing anti-TOPO-1 and/or anti-CENP-B autoantibodies in the sera. However, the cause-effect relationship between the two autoantibodies and RP remains elucidation. Sera containing anti-CENP-B and anti-TOPO-1 autoantibodies were obtained from SSc-related diseases manifesting RP. The polyclonal auto-antibodies were purified from pooled sera by affinity chromatography. Mouse monoclonal anti-CENP-B and anti-TOPO-1 were purchased. Calf pulmonary arterial endothelial cells (CPAE) were incubated with 40% patient sera, purified polyclonal antibodies or mouse monoclonal antibodies for 1-6 days. The vascular endothelial biomarkers von Willebrand factor (vWF), thrombomodulin (CD141) and 6-keto-prostaglandin F1α (6-keto-PGF1α), cell viability marker ATP, and cell necrosis/lysis marker LDH in the culture supernatants were measured by ELISA. The cell senescence biomarker β-galactosidase and telomere content in the cells were stained by the respective kit. The classical p53-p21 senescence pathway was detected by Western blot. We found that 40% anti-CENP-B or anti-TOPO-1-containing sera without heat-inactivation and mouse monoclonal antibodies suppressed 6-keto-PGF1α production, increased β-galactosidase, and decreased relative telomere content. The cell senescence effects were proved not via p53-p21 pathway. The pathognomonic anti-CENP-B and anti-TOPO-1 autoantibodies in SSc-related diseases accelerate vascular endothelial cell senescence and functional impairment inducing RP. The real signaling pathway for autoantibody-induced cell senescence remains exploration.

The arrangement of microvessels in human bone marrow is so far unknown. We combined monoclonal antibodies against CD34 and against CD141 to visualise all microvessel endothelia in 21 serial sections of about 1 cm2 size derived from a human iliac crest. The specimen was not decalcified and embedded in Technovit® 9100. In different regions of interest, the microvasculature was reconstructed in three dimensions using automatic methods. The three-dimensional models were subject to a rigid semiautomatic and manual quality control. In iliac crest bone marrow, the adipose tissue harbours irregularly distributed haematopoietic areas. These are fed by networks of large sinuses, which are loosely connected to networks of small capillaries prevailing in areas of pure adipose tissue. Our findings are compatible with the hypothesis that capillaries and sinuses in human iliac crest bone marrow are partially arranged in parallel.

BACKGROUND

Vitamin D3 (VD3) has been described as a modulator of immune system cells, including dendritic cells (DC). Previous studies have shown its importance in in vitro generation of tolerogenic DC, which have a similar function and phenotype to that of CD141 dermal DCs that produce IL-10 and induce (LTreg) CD4+ T regulator cells.

OBJECTIVE

This paper presents a study that compares the phenotype and cytokines produced by DC generated in presence and absence of VD3, which were matured with lipopolysaccharide (LPS), and their ability to induce LTreg from naïve allogeneic CD4+ T cells.

METHODS

In order to compare them, peripheral blood mononuclear cells were isolated to select monocytes CD14+ T cells and differentiate them in vitro from DC in the presence and absence of VD3, and to mature them with LPS. Phenotype and cytokine levels were also analyzed in the culture supernatants. Dendritic cells were then co-cultured with naïve allogeneic CD4+ T cells and the frequencies of LTreg were determined (naïve-activated).

RESULTS

The results showed that unstimulated DC generated with VD3 kept the CD14. When activated with LPS, they expressed lower levels of C83, CD83 and CD86; HLA-DR; higher amounts of IL-1β, IL-8, IL-10, and tended to lessen IL-6, IL-12p70 and TGF-β1, compared to DCs not treated with VD3. The frequency of naïve LTreg was similar, although immature DC generated with VD3 tended to induce activated LTregs.

CONCLUSIONS

Based on these results, it is possible to conclude that DCs generated with VD3 and treated with LPS presented a 'semi-mature' phenotype, and were able to secrete pro-inflammatory and anti-inflammatory cytokines. Besides, they did not increase their capacity to promote the polarization of naïve allogenic CD4+ T cells towards LTregs.

CD1c+ and CD141+ dendritic cells cooperate to sustain antiviral T cell responses.

Altered expression of vascular endothelial cell (EC) surface antigens in response to irradiation is one of the early events of radiation-induced damage. Using flow cytometry, we investigated the immunocytochemical reactivity of a blind panel comprising 87 mAbs submitted to the endothelial section of the 6th International Workshop on Human Leukocyte Differentiation Antigens with irradiated and resting human dermal microvascular endothelial (HDME) and EA cell lines. Monolayers of irradiated cells received a single 5 Gy dose of 72 h prior to staining but were otherwise treated the same as resting cells. For comparative purposes we have also examined the immunohistochemical reactivity of the mAb panel with EC in ovarian tumour, Wilms' tumour and human placenta. In the flow cytometry experiments 42 and 44 mAbs stained HDME and EA cells respectively and while no antibody stained irradiated but not unirradiated cells, upregulation was seen for CD31, CD34, CD141 and CD146 in irradiated cells. The upregulation of thrombomodulin (CD141) is noteworthy since it is a marker of EC damage and thus may be a useful reagent in investigations of vascular injury. Comparison with tissue staining showed that 21 mAbs were reactive with at least one tissue but not with either EA or HDME cells. Nine mAbs showed no cross reactivity with tissue and of these one reacted with EA cells only.

The lack of suitable animal models for the study of cytoadhesion of P. falciparum-infected erythrocytes (IEs) has necessitated in vitro studies employing a range of cell lines of either human tumour origin (e.g., BeWo and C32 cells) or non-human origin (e.g., CHO cells). Of the human cells available, many were isolated from adults, or derived from a pool of donors (e.g., HBEC-5i). Here we demonstrate, for the first time, the successful isolation of blood outgrowth endothelial cells (BOECs) from frozen stabilates of peripheral blood mononuclear cells obtained from small-volume peripheral blood samples from paediatric malaria patients. BOECs are a sub-population of human endothelial cells, found within the peripheral blood. We demonstrate that these cells express receptors such as Intercellular Adhesion Molecule 1 (ICAM-1/CD54), Endothelial Protein C Receptor (EPCR/CD201), platelet/endothelial cell adhesion molecule 1 (PECAM-1/CD31), Thrombomodulin (CD141), and support adhesion of P. falciparum IEs.

Myeloid malignancies showing CD141+ myeloid dendritic cell (MDC) differentiation have not been documented. Here, we describe a patient with juvenile myelomonocytic leukemia in which a prominent CD141+ cell population was identified most consistent with CD141+ MDCs based on phenotypic similarity with normal CD141+ MDCs. Molecular studies demonstrated a KRAS mutation. The findings from the spleen and bone marrow are described. This is the first well-documented demonstration of CD141+ MDC differentiation of a hematopoietic neoplasm.

CD93 belongs to the group XIV C-type lectin like domain (CTLD) and is closely related to thrombomodulin (CD141). Although CD93 is known to be involved in the regulation of cell adhesion and phagocytosis, its role in innate immunity remains to be fully investigated. Critically, published data about CD141 suggest that CD93 CTLD could be involved in the control of inflammation. In order to address further functional and structural analyses, we expressed human CD93 CTLD with several disulfide bonds in an E. coli expression system. As the E. coli cytoplasm is a reducing compartment, production of disulfide-bond proteins remains a challenge. Hence, we decided to over express CD93 CTLD in commercially available strains of E. coli and co-expressed a sulfhydryl oxidase (Erv1p) and a disulfide isomerase (DsbC). This strategy led to high yield expression of a native form of CD93 CTLD. NMR studies revealed that Ca2+ was not able to bind to CD93 CTLD. We also showed that the recombinant protein could alter LPS pro-inflammatory activity on THP1. This work provides new tool for further functional and structural studies to decipher the functions associated to the CTLD of CD93. This approach may also be used for others members of the group XIV C-type lectin like domain (CD141, CD248 and CLec14A).

Humanized mice have become useful animal models for HIV/AIDS. Since NOD.Cg-Prkdc scid Il2rgtm1Wjl/SzJ (NSG) mice allow the engraftment of primary human immune cells, we aim to determine the role of human Fms-related tyrosine kinase 3 ligand (hFlt3L), a major growth factor for dendritic cells (DCs), in regulating the differentiation of cord blood-derived CD34+ progenitor cells in this murine species. Soluble recombinant hFlt3L protein and AAV-vectored hFlt3L were administrated before or after human CD34+ progenitor cell transplantation, respectively. We then measured the peripheral levels of hFlt3L by ELISA. Meantime, reconstituted human immune cells were analyzed by flow cytometry over time. We found that without hFlt3L there were significantly increased types of human immune cells in NSG-huCD34 compared with NSG-huPBL mice but the frequency of human DCs remains low. Transient treatment with recombinant hFlt3L expanded human conventional CD1c+ and CD141+ DCs as well as plasmacytoid DCs in humanized NSG-huCD34 mice. Surprisingly, however, the prolonged in vivo expression of AAV-vectored hFlt3L resulted in significant suppression of total human CD34+ cell engraftment and differentiation. The suppression occurred within 2 weeks when AAV-vectored hFlt3L was administered either before or after the transplantation of CD34+ progenitor cells, which was likely associated with the induction of murine myeloid-derived immune suppressive cells and reactive oxygen species in NSG-huCD34 mice. Since chronic HIV-1 patients displayed significantly high levels of hFlt3L expression, our findings may have implication to explore the role of prolonged hFlt3L in regulating the differentiation of human CD34+ progenitor cells in both NSG-huCD34 mice and infected people. Graphical Abstract ᅟ.

Thrombomodulin is expressed on endothelial cells and monocytes (mTM) where it has an anticoagulant function. Enzymatic cleavage from the cell surface produces soluble thrombomodulin (sTM) in plasma. Abnormal levels of sTM and mutations in the thrombomodulin gene (THBD) are linked to cardiovascular disease. The aim of this study was to investigate THBD proximal promoter mutations and levels of sTM and mTM in men presenting with premature acute coronary syndrome (ACS). This prospective cross-sectional study included 100 adult men with premature ACS (age <55 years) and 60 healthy age-matched controls. Plasma sTM was assayed by ELISA. mTM expression was assessed by flow cytometry with CD141 antibody. The -33 G/A polymorphism was identified by PCR-restriction fragment length polymorphism analysis and the THBD proximal promoter region was sequenced. Significantly lower sTM (P < 0.001) and higher mTM (P < 0.001) were seen in ACS patients. Heterozygous THBD promoter polymorphisms -33 G/A and -9/-10 GG/AT were found in eight patients and five control individuals. In patients and control individuals, allele frequencies of A were 0.02 and 0.025, and that of AT were 0.025 and 0.017, respectively. There were no significant associations of these polymorphisms with ACS, sTM levels or mTM expression. THBD polymorphisms -33 G/A and -9/-10 GG/AT are present in low frequency in our patient population, and are more frequent in the South Asians as compared to the Arabs. The frequency of -33 G/A is lower, whereas that of -9/-10 GG/AT is higher than that reported in the Orientals. The presence of THBD proximal promoter polymorphisms do not explain variations in levels of sTM and mTM in this patient population.

Active co-delivery of tumor antigens (Ag) and α-galactosylceramide (α-GalCer), a potent agonist for invariant Natural Killer T (iNKT) cells, to cross-priming CD8α⁺ dendritic cells (DCs) was previously shown to promote strong anti-tumor responses in mice. Here, we designed a nanoparticle-based vaccine able to target human CD141⁺ (BDCA3⁺) DCs - the equivalent of murine CD8α⁺ DCs - and deliver both tumor Ag (Melan A) and α-GalCer. This nanovaccine was inoculated into humanized mice that mimic the human immune system (HIS) and possess functional iNKT cells and CD8⁺ T cells, called HIS-CD8/NKT mice. We found that multiple immunizations of HIS-CD8/NKT mice with the nanovaccine resulted in the activation and/or expansion of human CD141⁺ DCs and iNKT cells and ultimately elicited a potent Melan-A-specific CD8⁺ T cell response, as determined by tetramer staining and ELISpot assay. Single-cell proteomics further detailed the highly polyfunctional CD8⁺ T cells induced by the nanovaccine and revealed their predictive potential for vaccine potency. This finding demonstrates for the first time the unique ability of human iNKT cells to license cross-priming DCs in vivo and adds a new dimension to the current strategy of cancer vaccine development.

Keywords: CD8+ T cells; human CD141+ DCs; human immune system mice; iNKT cells; melanoma; nanovaccine; targeting; α-galactosylceramide.

Study question: What are the detailed endometrial tissue specific and systemic dendritic cell (DC) subset disturbances in endometriosis?

Summary answer: This study confirms myeloid DC (mDC) and plasmacytoid DC subsets are readily identified in endometrial tissue and shows both endometrial and circulating differences in DC populations in women with endometriosis, with disease stage-specific relationships evident locally in the endometrium.

What is known already: Immune factors in the uterus, the peritoneal environment and systemically are implicated in the pathogenesis and progression of both endometriosis and infertility. While there is some evidence that endometrial DC populations are altered in endometriosis, DC subset involvement in both the endometrium and peripheral blood have not been comprehensively investigated so the functional consequences have been unknown.

Study design, size, duration: This prospective cross-sectional cohort study compares circulating and endometrial DC populations in women of reproductive age with and without endometriosis (n = 55 and 30, respectively), wherein each participant donated samples at a single time point. Study participants were surveyed for menstrual cycle phase, American Society for Reproductive Medicine (ASRM) endometriosis disease stage and fertility status (where possible).

Participants/materials, setting, methods: Peripheral blood samples were processed into mononuclear cells for analysis by flow cytometry, and endometrial samples were analysed by immunohistochemistry and dissociated into single-cell suspension for flow cytometry.

Main results and the role of chance: In the endometrium of women with endometriosis, IRF-8+ cells were increased during the proliferative phase (P = 0.014), total DC proportions increased in the secretory phase (P = 0.038) and normal menstrual cyclical fluctuations in CD1c+ and IRF-8+ cells blunted; indicative of a consistently inflammatory tissue environment. The inflammatory changes in CD141+ and IRF-8+ populations in the endometrium of women with endometriosis were particularly evident in more advanced ASRM stages of the disease (respective P-values 0.032 and 0.045). There was also evidence of systemic inflammation in women with endometriosis, with increased circulating CD141+ mDC proportions (overall P = 0.040, secretory phase P = 0.021).

Large scale data: N/A.

Limitations, reasons for caution: As is common in this type of study, one of the main limitations was small sample numbers, particularly during the menstrual phase of the cycle.

Wider implications of the findings: Further phenotyping of local and circulating immune cell subtypes is critical to improving understanding of endometriosis pathogenesis and immune contributions to infertility associated with the disease.

Study funding/competing interest(s): This research was financially supported by a Sydney Medical School and Balnaves Foundation Kick Start Grant and the Department of Obstetrics, Gynaecology and Neonatology at The University of Sydney. The authors have no conflicts of interest to declare.

Keywords: blood; dendritic cells; endometriosis; endometrium; flow cytometry; immunohistochemistry; inflammation; menstrual cycle.

Background: We previously reported CpG-B injection at the primary tumor excision site prior to re-excision and sentinel node biopsy to result in immune activation of the sentinel lymph node (SLN), increased melanoma-specific CD8⁺ T cell rates in peripheral blood, and prolonged recurrence-free survival. Here, we assessed recruitment and activation of antigen-presenting cell (APC) subsets in the SLN and at the injection site in relation to T cell infiltration.

Methods: Re-excision skin specimens from patients with clinical stage I-II melanoma, collected 7 days after intradermal injection of either saline (n=10) or 8 mg CpG-B (CPG7909, n=12), were examined by immunohistochemistry, quantifying immune subsets in the epidermis, papillary, and reticular dermis. Counts were related to flow cytometric data from matched SLN samples. Additional in vitro cultures and transcriptional analyses on peripheral blood mononuclear cells (PBMCs) were performed to ascertain CpG-induced APC activation and chemokine profiles.

Results: Significant increases in CD83⁺, CD14⁺, CD68⁺, and CD123⁺ APC were observed in the reticular dermis of CpG-B-injected skin samples. Fluorescent double/triple staining revealed recruitment of both CD123⁺BDCA2⁺ plasmacytoid dendritic cells (DCs) and BDCA3/CD141⁺CLEC9A⁺ type-1 conventional DC (cDC1), of which only the cDC1 showed considerable levels of CD83 expression. Simultaneous CpG-B-induced increases in T cell infiltration were strongly correlated with both cDC1 and CD14 counts. Moreover, cDC1 and CD14⁺ APC rates in the reticular dermis and matched SLN suspensions were positively correlated. Flow cytometric, transcriptional, and chemokine release analyses of PBMC, on in vitro or in vivo exposure to CpG-B, indicate a role for the activation and recruitment of both cDC1 and CD14⁺ monocyte-derived APCs in the release of CXCL10 and subsequent T cell infiltration.

Conclusion: The CpG-B-induced concerted recruitment of cDC1 and CD14⁺ APC to the injection site and its draining lymph nodes may allow for both the (cross-)priming of T cells and their subsequent homing to effector sites.

Keywords: Immunotherapy; dendritic cells; lymphocytes; melanoma; translational medical research; tumor-infiltrating.

Allogeneic stem cell transplantation (alloSCT), following induction chemotherapy, can be curative for hemato-oncology patients due to powerful graft-versus-tumor immunity. However, disease recurrence remains the major cause of treatment failure, emphasizing the need for potent adjuvant immunotherapy. In this regard, dendritic cell (DC) vaccination is highly attractive, as DCs are the key orchestrators of innate and adaptive immunity. Natural DC subsets are postulated to be more powerful compared with monocyte-derived DCs, due to their unique functional properties and cross-talk capacity. Yet, obtaining sufficient numbers of natural DCs, particularly type 1 conventional DCs (cDC1s), is challenging due to low frequencies in human blood. We developed a clinically applicable culture protocol using donor-derived G-CSF mobilized CD34⁺ hematopoietic progenitor cells (HPCs) for simultaneous generation of high numbers of cDC1s, cDC2s and plasmacytoid DCs (pDCs). Transcriptomic analyses demonstrated that these ex vivo-generated DCs highly resemble their in vivo blood counterparts. In more detail, we demonstrated that the CD141⁺CLEG9A⁺ cDC1 subset exhibited key features of in vivo cDC1s, reflected by high expression of co-stimulatory molecules and release of IL-12p70 and TNF-α. Furthermore, cDC1s efficiently primed alloreactive T cells, potently cross-presented long-peptides and boosted expansion of minor histocompatibility antigen-experienced T cells. Moreover, they strongly enhanced NK cell activation, degranulation and anti-leukemic reactivity. Together, we developed a robust culture protocol to generate highly functional blood DC subsets for in vivo application as tailored adjuvant immunotherapy to boost innate and adaptive anti-tumor immunity in alloSCT patients.

Keywords: CD34+ hematopoietic progenitor cells; Clinical application; Dendritic cells; NK cells; T cells; Vaccination.

Increased life expectancy is usually associated with comorbidities, such as cardio and cerebrovascular disease causing impaired functionality. A common underlying cause of these comorbidities is vascular inflammation and injury. Elevated levels of circulating microvesicles (cMV), as a product of a hemostatic and inflammatory cell activation, could be direct mapping of an imbalanced hemostasis. In this manuscript, we aimed to investigate by liquid biopsy whether successful aging can be discriminated by cMV levels and phenotype. To this purpose, we included 135 community-dwelling octogenarians in a cross-sectional study. Successful aging was defined as good functional (Barthel Index > 90 points, and Lawton index score > 7/4 points for women and men, respectively) and cognitive status (Spanish version of the Mini-Mental State Examination -MEC- > 24 points) and no need for institutionalization. Total, annexin V positive (AV⁺), and AV^- cMV from different cell origins from the vascular compartment were phenotypically characterized and quantified from fasting plasma samples by flow cytometry. Successful aging was associated with lower plasma concentrations of total and AV⁺ CD141⁺/CD41⁺-CD61⁺, and PAC1⁺/AV⁺, CD141⁺/AV⁺, and CD36⁺/AV^- cMV. From these phenotypes, ROC curve analyses revealed that CD141⁺/AV⁺ and CD141⁺/CD41⁺-CD61⁺/AV⁺ endothelial- and platelet-derived cMV discriminate successful and non-successful aging with an AUC (95%CI) of 0.655 (0.551, 0.758), P = 0.005, and 0.638 (0.535, 0.741), P = 0.013, respectively. In conclusion, successful aging is associated with low levels of cMV released by endothelial cells and platelets, indicating lower endothelial cell inflammation and platelet activation. Our results contribute to the understanding of the link between unsuccessful aging, cognitive decline and vascular cell inflammatory disturbances.

Keywords: circulating microvesicles; cognitive decline; endothelial cells; inflammation; octogenarians; platelet activation; successful aging; thrombomodulin.

Dendritic cells (DCs) bridge the connection between innate and adaptive immunity. DCs present antigens to T cells and stimulate potent cytotoxic T cell responses. Metabolic reprogramming is critical for DC development and activation, however metabolic adaptations and regulation in DC subsets remains largely uncharacterized. Here, we mapped metabolomic and lipidomic signatures associated with the activation phenotype of human conventional DC type 1, a DC subset specialized in cross-presentation and therefore of major importance for the stimulation of CD8⁺ T cells. Our metabolomics and lipidomics analyses showed that Toll like receptor (TLR) stimulation altered glycerolipids and amino acids in cDC1. Poly I:C or pRNA stimulation reduced triglycerides and cholesterol esters, as well as various amino acids. Moreover, TLR stimulation reduced expression of glycolysis regulating genes and did not induce glycolysis. Conversely, cDC1 exhibited increased mitochondrial content, oxidative phosphorylation (OXPHOS) upon TLR3 or TLR7/8 stimulation. Our findings highlight the metabolic adaptations required for cDC1 maturation.

Keywords: BDCA3; Dendritic cell Activation; Lipid Mediators; Metabolomics.

Dendritic cells (DCs) with capacity of antigen cross-presentation are of key interest for immunotherapy against cancer as they can induce antigen-specific cytotoxic T lymphocyte (CTL) responses. This study describes frequencies of DC subtypes in human tonsils and lymph nodes, and phenotypic aspects that may be targeted by adjuvant measures. From human tonsils and neck lymph nodes, DCs were identified through flow cytometry, and subsets of plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) were investigated. Maturity status was assessed and surface receptors with CTL-promoting potentials were studied. CD123⁺ pDCs as well as CD1c⁺, CD141⁺, and CD1c^-CD141^- mDCs were detected in tonsils and lymph nodes. Both sites featured a similar presence of DC subsets, with CD123⁺ pDC being dominant and CD141⁺ mDCs least frequent. Based on CD80/CD86 expression, all DC subtypes featured a low degree of maturation. Expression of pattern recognition receptors (PRRs) CD206, CD207, DC-SIGN, TLR2, and TLR4, as well as the chemokine receptor XCR1, indicated DC subset-specific receptor profiles. We conclude that tonsils and lymph nodes share common features in terms of DC subset frequency and maturation as well as PRR and XCR1 expression pattern. Our work suggests that both sites may be considered for vaccine deposition in DC-mediated immunotherapy.

Keywords: Cancer; Dendritic cell; Immunotherapy; PRR; Vaccination.