A Potent Leukocyte Transmigration Blocker: GT-73 Showed a Protective Effect against LPS-Induced ARDS in Mice.

We recently developed a molecule (GT-73) that blocked leukocyte transendothelial migration from blood to the peripheral tissues, supposedly by affecting the platelet endothelial cell adhesion molecule (PECAM-1) function. GT-73 was tested in an LPS-induced acute respiratory distress syndrome (ARDS) mouse model. The rationale for this is based on the finding that the mortality of COVID-19 patients is partly caused by ARDS induced by a massive migration of leukocytes to the lungs. In addition, the role of tert-butyl and methyl ester moieties in the biological effect of GT-73 was investigated. A human leukocyte, transendothelial migration assay was applied to validate the blocking effect of GT-73 derivatives. Finally, a mouse model of LPS-induced ARDS was used to evaluate the histological and biochemical effects of GT-73. The obtained results showed that GT-73 has a unique structure that is responsible for its biological activity; two of its chemical moieties (tert-butyl and a methyl ester) are critical for this effect. GT-73 is a prodrug, and its lipophilic tail covalently binds to PECAM-1 via Lys536. GT-73 significantly decreased the number of infiltrating leukocytes in the lungs and reduced the inflammation level. Finally, GT-73 reduced the levels of IL-1ß, IL-6, and MCP-1 in bronchoalveolar lavage fluid (BALF). In summary, we concluded that GT-73, a blocker of white blood cell transendothelial migration, has a favorable profile as a drug candidate for the treatment of ARDS in COVID-19 patients.

Naïve CD4+ T Cell Lymphopenia and Apoptosis in Chronic Hepatitis C Virus Infection Is Driven by the CD31+ Subset and Is Partially Normalized in Direct-Acting Antiviral Treated Persons.

Background: The mechanisms underlying naïve CD4+ lymphopenia during chronic Hepatitis C Virus (HCV) infection are unclear. Whether direct-acting antiviral (DAA) therapy restores peripheral naïve CD4+ T cell numbers and function is unknown. Methods: We enumerated frequencies and counts of peripheral naïve CD4+, CD4+CD31+ and CD4+CD31- T cells by flow cytometry in a cross sectional analysis comparing chronic HCV infected (n=34), DAA-treated(n=29), and age-range matched controls (n=25), as well as in a longitudinal cohort of HCV DAA treated persons (n=16). The cross-sectional cohort was stratified by cirrhosis state. Cell apoptosis/survival (AnnexinV+7AAD+/BCL-2 labeling) and cell cycle entry (Ki67 expression) of CD31+ and CD31- naïve CD4+ T cells was analyzed directly ex vivo and following 3 and 5 days of in vitro culture with media, interleukin (IL) -7 or CD3/CD28 activator. Results: In the cross-sectional cohort, naïve CD4+ proportions were lower in chronic HCV infected persons compared to controls and DAA-treated persons, an effect in part attributed to cirrhosis. Age was associated with naïve cell counts and proportions in HCV infected and treated persons as well. Naïve CD4+ cell proportions negatively correlated with plasma levels of soluble CD14 following therapy in DAA-treated persons. Naïve CD4+ cells from HCV infected persons exhibited greater direct ex vivo apoptosis and cell-cycling compared to cells from DAA-treated persons and controls, and this was localized to the CD4+CD31+ subset. On the other hand, no remarkable differences in expression of BCL-2 or IL-7 Receptor (CD127) at baseline or following in vitro media or IL7 containing culture were observed. In the longitudinal cohort, naïve CD4+CD31+/CD31- ratio tended to increase 24 weeks after DAA therapy initiation. Conclusions: Activation and apoptosis of peripheral naïve CD4+CD31+ T cells appear to contribute to naïve CD4+ lymphopenia in chronic HCV infection, and this defect is partially reversible with HCV DAA therapy. Age and cirrhosis -associated naïve CD4+ lymphopenia is present both before and after HCV DAA therapy. These findings have implications for restoration of host immune function after DAA therapy.

Rapid and efficient immunomagnetic isolation of endothelial cells from human peripheral nerves.

Endothelial cells (ECs) have gained an increased scientific focus since they were reported to provide guidance for Schwann cells and subsequently following axons after nerve injuries. However, previous protocols for the isolation of nerve-derived ECs from human nerves are ineffective regarding time and yield. Therefore, we established a novel and efficient protocol for the isolation of ECs from human peripheral nerves by means of immunomagnetic CD31-antibody conjugated Dynabeads and assessed the purity of the isolated cells. The easy-to-follow and time-effective isolation method allows the isolation of > 95% pure ECs. The isolated ECs were shown to express highly specific EC marker proteins and revealed functional properties by formation of CD31 and VE-cadherin positive adherens junctions, as well as ZO-1 positive tight-junctions. Moreover, the formation of capillary EC-tubes was observed in-vitro. The novel protocol for the isolation of human nerve-derived ECs allows and simplifies the usage of ECs in research of the human blood-nerve-barrier and peripheral nerve regeneration. Additionally, a potential experimental application of patient-derived nerve ECs in the in-vitro vascularization of artificial nerve grafts is feasible.

Multiple combinations of melanocytic and vascular endothelial markers enhance the detection rate of lymphovascular invasion in cutaneous melanoma.

BACKGROUND: Lymphovascular invasion (LVI) is believed to be the mechanism by which melanoma cells can disseminate to regional lymph nodes and distant sites and may be predictive of adverse outcome. Lymphovascular invasion often difficult to detect on hematoxylin-eosin (HE) stained sections, are readily identified with dual immunohistochemistry (IHC) for melanocytic and vascular markers. METHODS: A total of 100 primary cutaneous malignant melanoma cases that had a Breslow thickness of 1-4 mm and lacked LVI by conventional HE assessment were included. We compared the LVI detection rates of double staining for CD31/S100 and CD34/S100, and D2-40/S100, and examined the association of LVI with clinical outcomes. RESULTS: The dual immunohistochemical positivity for CD31/S100, CD34/S100, and D2-40/S100 were 40(40%), 17(17%) and 35(35%), respectively. On multivariate analysis, LVI was an independent predictor of SLN status. Multivariate analysis revealed that LVI and male gender were independent risk factors for overall survival. CONCLUSIONS: The recognition of LVI is improved by dual IHC and predicts SLN metastasis. The detection of LVI using dual IHC, especially by a combination of CD31/S100 and D2-40/S100 is a useful step that inclusion should be recommended in basic evaluation parameters for cutaneous melanoma.

Bivalent engagement of endothelial surface antigens is critical to prolonged surface targeting and protein delivery in vivo.

Targeted drug delivery to the endothelium has the potential to generate localized therapeutic effects at the blood-tissue interface. For some therapeutic cargoes, it is essential to maintain contact with the bloodstream to exert protective effects. The pharmacokinetics (PK) of endothelial surface-targeted affinity ligands and biotherapeutic cargo remain a largely unexplored area, despite obvious translational implications for this strategy. To bridge this gap, we site-specifically radiolabeled mono- (scFv) and bivalent (mAb) affinity ligands specific for the endothelial cell adhesion molecules, PECAM-1 (CD31) and ICAM-1 (CD54). Radiotracing revealed similar lung biodistribution at 30 minutes post-injection (79.3% ± 4.2% vs 80.4% ± 10.6% ID/g for αICAM and 58.9% ± 3.6% ID/g vs. 47.7% ± 5.8% ID/g for αPECAM mAb vs. scFv), but marked differences in organ residence time, with antibodies demonstrating an order of magnitude greater area under the lung concentration vs. time curve (AUCinf 1698 ± 352 vs. 53.3 ± 7.9 ID/g*hrs for αICAM and 1023 ± 507 vs. 114 ± 37 ID/g*hrs for αPECAM mAb vs scFv). A physiologically based pharmacokinetic model, fit to and validated using these data, indicated contributions from both superior binding characteristics and prolonged circulation time supporting multiple binding-detachment cycles. We tested the ability of each affinity ligand to deliver a prototypical surface cargo, thrombomodulin (TM), using one-to-one protein conjugates. Bivalent mAb-TM was superior to monovalent scFv-TM in both pulmonary targeting and lung residence time (AUCinf 141 ± 3.2 vs 12.4 ± 4.2 ID/g*hrs for ICAM and 188 ± 90 vs 34.7 ± 19.9 ID/g*hrs for PECAM), despite having similar blood PK, indicating that binding strength is more important parameter than the kinetics of binding. To maximize bivalent target engagement, we synthesized an oriented, end-to-end anti-ICAM mAb-TM conjugate and found that this therapeutic had the best lung residence time (AUCinf 253 ± 18 ID/g*hrs) of all TM modalities. These observations have implications not only for the delivery of TM, but also potentially all therapeutics targeted to the endothelial surface.

Dark-field microscopy enhance visibility of CD31 endothelial staining.

A simple dark field microscopy technique was used for visualization of blood vessels in normal human renal tissues and carcinoma. Phase contrast condenser ring apt for high power objectives was combined with a 10x objective in order to create a dark field illumination of the specimens examined. The endothelial lining of the vessels had been stained by using CD31 monoclonal antibodies combined with conventional peroxidase immunohistochemistry. The final DAB addition used for this technique induced an intense light scatter in the dark field microscope. This scattered light originating from the endothelial lining made the walls of the bright vessels easily detectable from the dark background.

Human CD31 on Swine Endothelial Cells Induces SHP-1 Phosphorylation in Macrophages.

BACKGROUND: Innate immunity by natural killer (NK) cells, macrophages, and neutrophils cause severe rejections in xenotransplantation. Therefore, the development of strategies for suppressing macrophages has considerable potential in practical applications of xenotransplantation. Recently, we found that human CD31 on swine endothelial cells (SECs) suppresses neutrophil-mediated xenogeneic rejection through homophilic binding. Since a significant amount of CD31 is expressed not only on neutrophils but also on macrophages, we studied the function of human CD31 in macrophage-mediated cytotoxicity. METHODS: SECs and hCD31-transfected SECs (SEC/hCD31) were co-cultured with macrophages and cytotoxicity by macrophages was evaluated with water-soluble tetrazolium salt, or WST-8, assay. To confirm whether or not inhibitory signals are induced by hCD31 homophilic binding, the phosphorylation of the enzyme SHP-1 was investigated with Western blotting. RESULTS: No suppression of cytotoxicity was induced in macrophages that had been co-cultured with SEC/CD31. However, phosphorylation of SHP-1 was induced in macrophages that had been co-cultured with SEC/hCD31. CONCLUSIONS: Human CD31 on SEC may induce not only inhibitory signals but also activation signals via the binding to other receptors for hCD31.

Associations between recent thymic emigrants and CD4+ T-cell recovery after short-term antiretroviral therapy initiation.

OBJECTIVE: Around 20-30% of HIV-infected individuals (HIV+) on successful antiretroviral therapy (ART) fail to normalize their CD4 T-cell counts. Various factors could contribute to the lack of immune reconstitution, one of them being thymic insufficiency. We aimed to explore associations between recent thymic emigrants (RTEs) and CD4 T-cell recovery. DESIGN: ART-naive HIV+ individuals who started ART with advanced AIDS were selected. Good versus poor immune reconstitution was defined by CD4 gains above or below 100 CD4 T cells/µl. The follow-up period was 6 months. METHODS: Peripheral blood mononuclear cells were isolated and flow cytometry was used to characterize RTEs as the fraction of naive CD4 T cells expressing CD31, the platelet endothelial cell adhesion molecule. Markers of cellular activation, senescence, exhaustion and cycling were also assessed. RESULTS: After 6 months on ART, HIV+ individuals with good immune reconstitution had higher absolute numbers of RTEs, compared with those with poor immune reconstitution, and these strongly correlated with CD4 gains in those individuals with good immune reconstitution but not with poor immune reconstitution. We also found that CD8 T-cell immune activation decreased as early as 2 months post-ART initiation in individuals with good immune reconstitution, but only at month 6 post-ART in individuals with poor immune reconstitution. Levels of immune activation were inversely correlated with the absolute numbers of RTEs in both groups, but more strongly so in individuals with poor immune reconstitution. CONCLUSION: We show that RTEs are linked to CD4 T-cell recovery and that the degree of immune reconstitution is not directly linked to persistent immune activation.

Cell generation dynamics underlying naive T-cell homeostasis in adult humans.

Thymic involution and proliferation of naive T cells both contribute to shaping the naive T-cell repertoire as humans age, but a clear understanding of the roles of each throughout a human life span has been difficult to determine. By measuring nuclear bomb test-derived 14C in genomic DNA, we determined the turnover rates of CD4+ and CD8+ naive T-cell populations and defined their dynamics in healthy individuals ranging from 20 to 65 years of age. We demonstrate that naive T-cell generation decreases with age because of a combination of declining peripheral division and thymic production during adulthood. Concomitant decline in T-cell loss compensates for decreased generation rates. We investigated putative mechanisms underlying age-related changes in homeostatic regulation of CD4+ naive T-cell turnover, using mass cytometry to profile candidate signaling pathways involved in T-cell activation and proliferation relative to CD31 expression, a marker of thymic proximity for the CD4+ naive T-cell population. We show that basal nuclear factor κB (NF-κB) phosphorylation positively correlated with CD31 expression and thus is decreased in peripherally expanded naive T-cell clones. Functionally, we found that NF-κB signaling was essential for naive T-cell proliferation to the homeostatic growth factor interleukin (IL)-7, and reduced NF-κB phosphorylation in CD4+CD31- naive T cells is linked to reduced homeostatic proliferation potential. Our results reveal an age-related decline in naive T-cell turnover as a putative regulator of naive T-cell diversity and identify a molecular pathway that restricts proliferation of peripherally expanded naive T-cell clones that accumulate with age.

The vasa vasorum reach deep into the human thoracic aorta.

Only limited data are available on the extent of the vasa vasorum of the human thoracic aorta, although this could be important with regard to certain pathophysiological states, i.e. aortic aneurysm or atherosclerosis. A preliminary investigation shows that the vascularization of the human thoracic aorta reaches deeper layers than generally believed.

Anatomy, immunohistochemistry, and numerical distribution of human splenic microvessels.

The microvascular architecture of the spleen plays an important role in the immunological function of this organ. The different types of vessels are related to different reticular cells each with their own immunomodulatory functions. The present study describes an immunohistochemical and morphometric analysis of the various types of vessels in 21 human autopsy non-pathological splenic samples. On an area of 785,656.37 µm2 for each sample, we classified and quantified the type and number of vascular structures, each according to their morphology and immunohistochemical profile, and obtained the ratios between them. The distribution of trabecular vessels and the characteristics of the venules are reviewed. In our material the so-called "cavernous perimarginal sinus" (anatomical structure previously described by Schmidt et al., 1988) was observed and interpreted as a curvilinear venule shaped by the follicle in contact with the trabecular vein. Our material comprised 261 trabeculae (containing 269 arterial sections and 508 venous sections), 30,621 CD34+ capillaries, 7739 CD271+ sheathed capillaries, 2588 CD169+ sheathed capillaries, and 31,124 CD8+ sinusoids. The total area (TA) (14,765,714.88 µm2) occupied by the sinusoidal sections of the 21 cases was much higher than the TA of the capillary sections (1,700,269.83 µm2). Similarly, the TA (651,985 µm2) occupied by the sections of the trabecular veins was much higher than the TA of the trabecular arteries (88,594 µm2). The total number of CD34+ capillaries and of sinusoids CD8+ was similar for the sum of the 21 cases, nevertheless there were large differences in each case. Statistically the hypothesis that the number of capillaries and sinusoids are present with the same frequency is discarded. In view of the absence of a numerical correlation between capillaries and sinusoids, we postulate that very possibly the arterial and the venous vascular trees are two anatomically independent structures separated by the splenic cords. We believe that this is the first work where splenic microvascularization is simultaneously approached from a morphometric and immunohistochemical point of view.

Mechanotransduction, immunoregulation, and metabolic functions of CD31 in cardiovascular pathophysiology.

Biomechanical changes in the heart and vessels drive rapid and dynamic regulation of blood flow, a vital process for meeting the changing metabolic needs of the peripheral tissues at any given point in time. The fluid movement of the blood exerts haemodynamic stress upon the solid elements of the cardiovascular system: the heart, vessels, and cellular components of the blood. Cardiovascular diseases can lead to prolonged mechanical stress, such as cardiac remodelling during heart failure or vascular stiffening in atherosclerosis. This can lead to a significantly reduced or increasingly turbulent blood supply, inducing a shift in cellular metabolism that, amongst other effects, can trigger the release of reactive oxygen species and initiate a self-perpetuating cycle of inflammation and oxidative stress. CD31 is the most abundant constitutive co-signalling receptor glycoprotein on endothelial cells, which line the cardiovascular system and form the first-line of cellular contact with the blood. By associating with most endothelial receptors involved in mechanosensing, CD31 regulates the response to biomechanical stimuli. In addition, by relocating in the lipid rafts of endothelial cells as well as of cells stably interacting with the endothelium, including leucocytes and platelets, CD31-CD31 trans-homophilic engagement guides and restrains platelet and immune cell accumulation and activation and at sites of damage. In this way, CD31 is at the centre of mediating mechanical, metabolic, and immunological changes within the circulation and provides a single target that may have pleiotropic beneficial effects.

1,25-Dihydroxyvitamin D3 Restrains CD4+ T Cell Priming Ability of CD11c+ Dendritic Cells by Upregulating Expression of CD31.

Dendritic cells (DC) are specialized sentinel cells that bridge the innate and adaptive immune response and play a crucial role in shaping the adaptive immune response. Vitamin D, a known epidemiological risk factor for the development of several autoimmune diseases, influences the development of dendritic cells. Consequently, vitamin D metabolites are frequently used in protocols to develop therapeutic dendritic cell therapies for autoimmune diseases. However, the mechanisms by which vitamin D modulates DC function remain poorly understood. We investigated the effects of vitamin D on murine CD11c+ bone marrow derived DC (BMDC) function by analyzing global gene expression in CD11c+ BMDC generated in the presence (VitD-CD11c+BMDC) or absence (Veh-CD11c+BMDC) of the active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Seven genes were significantly increased in expression in both immature and LPS-matured VitD-CD11c+BMDC, one of which was CD31, a member of the immunoglobulin superfamily. Gene knockdown of CD31 enhanced the ability of VitD-CD11c+BMDC to prime naïve CD4+ T cells in vitro; conversely, increased expression of CD31 on vehicle treated CD11c+BMDC restrained their T cell priming abilities. Time-lapse imaging of BMDC and CD4+ T cells during in vitro priming revealed that CD31 reduced the BMDC-T cell interaction time. Finally, we confirmed a similar effect of 1,25(OH)2D3 on human CD34+ cell-derived CD11c+DC, whereby DC generated in the presence of 1,25(OH)2D3 had increased CD31 expression. In summary, we show that both mouse and human DC generated in the presence of 1,25(OH)2D3 upregulate CD31 expression, resulting in a reduced ability to prime CD4+ T cells by impairing a stable cell-cell contact.

Molecular mechanisms of immunoreceptors in platelets.

BACKGROUND: The main role of platelets is to control haemostasis when there is a blood vessel injury in order to minimise blood loss at the injury site. Under normal circumstances, platelets flow freely within blood vessels as the endothelial cells provide a non-adhesion surface. Naturally, bioactive mediators are released from endothelial cells to prevent and control platelet activation. However, when the vascular endothelium is ruptured, the local concentration of nitric oxide and prostaglandin is diminished and receptors containing a sequence of amino acids known as, immunoreceptor tyrosine-based inhibition motifs (ITIMs), serve as natural inhibitors within platelets. The main role of ITIMs is to decrease immunoreceptor tyrosine-based activation motif (ITAM) signalling in platelets; however, some studies have revealed their novel role in integrin αIIbß3 activation. This review highlights the main structural and functional features of immunoreceptors in platelets.

Thymic output changes in children with clinical findings signaling a probable primary immunodeficiency.

Karaca N, Azarsiz E, Akarcan SE, Aksu G, Kütükçüler N. Thymic output changes in children with clinical findings signaling a probable primary immunodeficiency. Turk J Pediatr 2019; 61: 885-894. Thymic maturation evaluation is inevitable for patients with clinical and laboratory findings for a primary immunodeficiency, as the T cellimmunodeficiencies are the most severe type. In this study, we aimed to show the usage of T cell surface molecule `CD31` for the evaluation of thymic output in patients (n: 66) with a large spectrum of findings signing a probable primary immunodeficiency. Besides the classical clinical and laboratory approach for these patients, T cell subpopulations as naive, memory, recent thymic emigrant cells were also investigated. The humoral immunodeficiency (34.8%), combined immunodeficiency (34.8%) and cardiopathy (7.6%) were the most frequent diagnosis groups. CD4+CD45RA+ naive T-cells percentages (p: 0.011) and absolute counts (p: 0.004) and absolute CD4+CD45RA+CD31+ RTE (recent thymic emigrant) cell counts (p: 0.007) were significantly lower in combined immunodeficiency group. Naive T-cells (p: 0.037) and RTE cells (p: 0.032) were also lower in patients who had cardiac surgery in the past. In conclusion, flow cytometric CD31+thymic naive RTE cell evaluation may provide rapid clinical information especially on T-cell immune dysfunction and CD4+CD45RA+CD31+ RTE cells may be used as an alternative to TRECs in the diagnosis of combined immunodeficiencies.

Isolation of tumor endothelial cells from murine cancer.

Tumor endothelial cells (TECs), which constitute the lining of the tumor blood vessels, have various characteristics as tumor constituent cells. In this study, we describe a novel method for the isolation of highly pure, fresh TECs, which form a small population within the tumor. Tumors were first dissected from tumor-bearing mice and digested to a single cell suspension with Collagenase Type II; the single cells were then separated by density gradient centrifugation. TECs were enriched by CD31-positive selection using magnetic activated cell sorting and subsequently purified by fluorescence activated cell sorting. The high purity of the obtained cells was verified by flow cytometry. Upon cell culture, the isolated cells showed a polygonal shape and a cobblestone appearance, which are features of the endothelial cells. Furthermore, a functional assay revealed that the TECs suppressed the proliferation of CD8+ T cells in vitro. We believe that the isolation method described in this study will enable the further elucidation of the characteristics of TECs.

Generation of an immortalized human choroid endothelial cell line (iChEC-1) using an endothelial cell specific promoter.

Age-related macular degeneration (AMD) is a common cause of blindness worldwide. While recent studies have revealed that the loss of choroidal endothelial cells (ChECs) is critical to the disease pathogenesis of dry AMD, in vitro studies are needed to fully elucidate the disease mechanism. However, these studies remain hindered due to the lack of publically available human ChEC lines. To address this need, ChECs were harvested form donor tissue and enriched for by using magnetic cell separation using anti-CD31 conjugated microbeads. Next, lenti-viral vectors with endothelial-specific promoters driving genes necessary for immortalization, CDH5p-hTERT and CDH5p TAg, were generated. Stable integration of both gene cassettes allowed cells to maintain their proliferative state and yielded an immortalized cell line (iChEC-1). Immunocytochemical analysis of iChEC-1 confirmed the expression of important ChEC markers such as CA4, a marker of choriocapillaris endothelial cells, CDH5, and CD34, pan-endothelial cell markers. qRT-PCR analysis of expanded clones from iChEC-1 further showed that the line maintained expression of other important endothelial markers, vWF, PECAM1, and PLVAP, similar to primary cells. Functional responses were characterized by tube-forming assays and repopulation of decellularized choroid with the immortalized cell line. In conclusion, the iChEC-1 line presents a suitable immortalized human ChEC line for future in vitro studies of AMD.

The Majority of CD45- Ter119- CD31- Bone Marrow Cell Fraction Is of Hematopoietic Origin and Contains Erythroid and Lymphoid Progenitors.

The non-hematopoietic cell fraction of the bone marrow (BM) is classically identified as CD45- Ter119- CD31- (herein referred to as triple-negative cells or TNCs). Although TNCs are believed to contain heterogeneous stromal cell populations, they remain poorly defined. Here we showed that the vast majority of TNCs (∼85%) have a hematopoietic rather than mesenchymal origin. Single cell RNA-sequencing revealed erythroid and lymphoid progenitor signatures among CD51- TNCs. Ly6D+ CD44+ CD51- TNCs phenotypically and functionally resembled CD45+ pro-B lymphoid cells, whereas Ly6D- CD44+ CD51- TNCs were enriched in previously unappreciated stromal-dependent erythroid progenitors hierarchically situated between preCFU-E and proerythroblasts. Upon adoptive transfer, CD44+ CD51- TNCs contributed to repopulate the B-lymphoid and erythroid compartments. CD44+ CD51- TNCs also expanded during phenylhydrazine-induced acute hemolysis or in a model of sickle cell anemia. These findings thus uncover physiologically relevant new classes of stromal-associated functional CD45- hematopoietic progenitors.

Frontline Science: PECAM-1 (CD31) expression in naïve and memory, but not acutely activated, CD8+ T cells.

Inhibitory cell surface proteins on T cells are often dynamically regulated, which contributes to their physiologic function. PECAM-1 (CD31) is an inhibitory receptor that facilitates TGF-ß-mediated suppression of T cell activity. It is well established in CD4+ T cells that PECAM-1 is expressed in naïve recent thymic emigrants, but is down-regulated after acute T cell activation and absent from memory cells. The extent to which PECAM-1 expression is similarly regulated in CD8+ T cells is much less well characterized. We evaluated T cells recovered from mice after infection with a model intracellular pathogen and determined that, in CD8+ T cells, PECAM-1 expression was strongly down-regulated during acute infection but re-expressed to intermediate levels in memory cells. Down-regulation of PECAM-1 expression in CD8+ T cells was transcriptionally regulated and affected by the strength and nature of TCR signaling. PECAM-1 was also detected on the surface of human activated/memory CD8+ , but not CD4+ T cells. These data demonstrate that PECAM-1 expression is dynamically regulated, albeit differently, in both CD4+ and CD8+ T cells. Furthermore, unlike memory CD4+ T cells, memory CD8+ T cells retain PECAM-1 expression and have the potential to be modulated by this inhibitory receptor.

Decreased expression of cell adhesion genes in cancer stem-like cells isolated from primary oral squamous cell carcinomas.

The goal of this study was to isolate cancer stem-like cells marked by high expression of CD44, a putative cancer stem cell marker, from primary oral squamous cell carcinomas and identify distinctive gene expression patterns in these cells. From 1 October 2013 to 4 September 2015, 76 stage III-IV primary oral squamous cell carcinoma of the gingivobuccal sulcus were resected. In all, 13 tumours were analysed by immunohistochemistry to visualise CD44-expressing cells. Expression of CD44 within The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma RNA-sequencing data was also assessed. Seventy resected tumours were dissociated into single cells and stained with antibodies to CD44 as well as CD45 and CD31 (together referred as Lineage/Lin). From 45 of these, CD44+Lin- and CD44-Lin- subpopulations were successfully isolated using fluorescence-activated cell sorting, and good-quality RNA was obtained from 14 such sorted pairs. Libraries from five pairs were sequenced and the results analysed using bioinformatics tools. Reverse transcription quantitative polymerase chain reaction was performed to experimentally validate the differential expression of selected candidate genes identified from the transcriptome sequencing in the same 5 and an additional 9 tumours. CD44 was expressed on the surface of poorly differentiated tumour cells, and within the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma samples, its messenger RNA levels were higher in tumours compared to normal. Transcriptomics revealed that 102 genes were upregulated and 85 genes were downregulated in CD44+Lin- compared to CD44-Lin- cells in at least 3 of the 5 tumours sequenced. The upregulated genes included those involved in immune regulation, while the downregulated genes were enriched for genes involved in cell adhesion. Decreased expression of PCDH18, MGP, SPARCL1 and KRTDAP was confirmed by reverse transcription quantitative polymerase chain reaction. Lower expression of the cell-cell adhesion molecule PCDH18 correlated with poorer overall survival in the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma data highlighting it as a potential negative prognostic factor in this cancer.