Immune Cell Subpopulations Research Articles

Increased Tumor Immune Microenvironment CD3+ and CD20+ Lymphocytes Predict a Better Prognosis in Oral Tongue Squamous Cell Carcinoma.

Background: Oral tongue squamous cell carcinoma (OTSCC) causes over 350,000 cases annually and particularly impacts populations in developing countries. Smoking and alcohol consumption are major risk factors. Determining the role of the tumor immune microenvironment (TIME) in OTSCC outcomes can elucidate immune mechanisms behind disease progression, and can potentially identify prognostic biomarkers.Methods: We performed a retrospective study of 48 OTSCC surgical specimens from patients with tobacco and alcohol exposures. A panel of immunoregulatory cell subpopulations including T (CD3, CD4, CD8) and B (CD20) lymphocytes, dendritic cells (CD1a, CD83), macrophages (CD68), and immune checkpoint molecules programmed cell death protein 1 (PD-1) and ligand 1 (PD-L1) were analyzed using immunohistochemistry. The levels of immune effector cell subpopulations and markers were analyzed in relation to overall survival.Results: Pathological characteristics of the tumor microenvironment included inflammatory infiltrates (83.3%), desmoplasia (41.6%), and perineural invasion (50.0%). The TIME contained high levels of T cells (CD3+, CD4+, and CD8+) and B cells (CD20+), as well as immature (CD1a) and mature (CD83) dendritic cells, PD-1, and PD-L1. Higher numbers of TIME infiltrating CD3+ T cells and CD20+ B cells were predictive of better survival, while higher levels of CD83+ mature dendritic cells predicted better survival. CD3+ T cells were identified as an independent prognostic marker for OTSCC. Lastly, CD3+ T cells were strongly correlated with the number of CD8+ cells and PD-L1 expression.Conclusion: Our findings provide evidence that the TIME profile of OTSSC impacted prognosis. The high expression of CD3+ T cells and B cells are predictive of better overall survival and indicative of an immunologically active, inflammatory TIME in patients with better survival. The number of CD3+ T cells was an independent prognostic marker.

Abstract PS18-27: Integrated immuno-genomic analyses in early breast cancer: Results from the Scandinavian breast group 2004-1 (SBG-2004-1) randomized phase II trial

Abstract Background: Given the emerging role of immune-related biomarkers in breast cancer (BC), little is known about specific immune cell composition patterns and genomic alterations and how these could interact in early BC setting. The aim of this proof-of-principle study is to describe immuno-genomic correlates within the tumor microenvironment using newly developed techniques for multiplexed fluorescent immunohistochemistry (mfIHC, Mezheyeuski et al., J Pathol. 2018) and high-throughput low-input genome sequencing (CUTseq, Zhang et al., Nat Commun 2019). Methods: The randomized SBG-2004-1 phase II trial evaluated the feasibility of tailored and dose-dense epirubicin and cyclophosphamide followed by docetaxel (EC→T) as adjuvant chemotherapy for early BC, enrolling a total of 124 patients. CUTseq was applied to gDNA extracted from archival low-input formalin-fixed paraffin-embedded (FFPE) tissue as well as peripheral blood samples of BC patients. mfIHC was performed on FFPE tissue microarrays, allowing simultaneous detection of six immune markers (CD4, CD8, PD-L1, PD-1, FoxP3, CD68) and quantification in an automated and tissue-compartment manner (Vectra® Polaris™ automated quantitative pathology imaging system and inForm® software, Akoya Biosciences). Patient characteristics and 10-year follow-up data were also available. Associations of different immune cell patterns and DNA copy number alterations (CNAs) with clinicopathological parameters and survival outcomes were evaluated using standard statistical methods.Results: 69/124 (55.6%) and 82/124 (66.1%) FFPE samples were available for CNA profiling (blood gDNA for normalization, n=33) and mfIHC, respectively. MYC gene was the most commonly amplified cancer-associated gene (n=44, 63.8%), and other frequent alterations included ERBB2 (n=31, 44.9%) and PIK3CA (n=14, 20.3%) amplification as well as TP53 (n=29, 42%) and PTEN (n=16, 23.2%) deletion. CNA burden (i.e., the percentage of the genome either amplified or deleted) was significantly correlated with higher tumor grade (p=0.013) but was not prognostic for relapse-free survival. CD4+ T-cells were the most abundant immune cell subpopulation, followed by CD68+ macrophages. The immune checkpoint markers PD-L1 and PD-1 were mostly expressed in CD4+ T-cells in both tumor and stroma compartments, while stromal PD-1+CD8+ and PD-1+CD68+ cell subsets were also among the ones with the higher cell density. CD4+ T-cell density was significantly correlated with tumor size, whereas the different immune cell infiltration patterns were not found to be prognostic. Combined immuno-genomic analyses revealed that high CNA burden was inversely associated with intra-tumoral CD4+ (Spearman’s r=-0.33, p=0.01) and CD8+ infiltration (Spearman’s r=-0.37, p=0.004). Conclusions: The present study indicates the feasibility of CNA profiling by CUTseq and immuno-profiling by mfIHC in FFPE samples obtained from BC patients. Moreover, our data provide a link between genomic alterations and the immune landscape in early BC and set the basis for further application of CUTseq and mfIHC in larger patient cohorts. Citation Format: Ioannis Zerdes, Michele Simonetti, Alexios Matikas, Luuk Harbers, Ning Zhang, Dimitrios Salgkamis, Susanne Agartz, Markella Zacharouli, Pablo Moreno-Ruiz, Johan Hartman, Artur Mezheyeuski, Jonas Bergh, Nicola Crosetto, Theodoros Foukakis. Integrated immuno-genomic analyses in early breast cancer: Results from the Scandinavian breast group 2004-1 (SBG-2004-1) randomized phase II trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-27.

Effects of cold water immersion on circulating inflammatory markers at the Kona Ironman World Championship.

Cold water immersion (CWI) purportedly reduces inflammation and improves muscle recovery after exercise, yet its effectiveness in specific contexts (ultraendurance) remains unclear. Thus, our aim was to study hematological profiles, systemic inflammation, and muscle damage responses to a specific post-race CWI (vs. control) during recovery after the Ironman World Championship, a culmination of ∼100 000 athletes competing in global qualifying Ironman events each year. Twenty-nine competitors were randomized into either a CWI or control (CON) group. Physiological parameters and blood samples were taken at pre-race, after intervention (POST), and 24 (+1DAY) and 48 hours (+2DAY) following the race. Muscle damage markers (plasma myoglobin, serum creatine kinase) were elevated at POST, +1DAY, and +2DAY, while inflammatory cytokines interleukin (IL)-6, IL-8, and IL-10 and total leukocyte counts were increased only at POST. CWI had no effect on these markers. Numbers of the most abundant circulating cell type, neutrophils, were elevated at POST more so in CWI (p < 0.05, vs. CON). Despite that neutrophil counts may be a sensitive marker to detect subtle effects, CWI does not affect recovery markers 24- and 48-hours post-race (vs. CON). Overall, we determined that our short CWI protocol was not sufficient to improve recovery. Novelty: Ironman World Championship event increased circulating muscle damage markers, inflammatory markers, and hematological parameters, including circulating immune cell sub-populations that recover 24-48 hours after the race. 12-min CWI post-ultraendurance event affects the absolute numbers of neutrophils acutely, post-race (vs. CON), but does not impact recovery 24- and 48-hours post-race.

Single-cell resolution landscape of equine peripheral blood mononuclear cells reveals diverse cell types including T-bet+ B cells

BackgroundTraditional laboratory model organisms represent a small fraction of the diversity of multicellular life, and findings in any given experimental model often do not translate to other species. Immunology research in non-traditional model organisms can be advantageous or even necessary, such as when studying host-pathogen interactions. However, such research presents multiple challenges, many stemming from an incomplete understanding of potentially species-specific immune cell types, frequencies, and phenotypes. Identifying and characterizing immune cells in such organisms is frequently limited by the availability of species-reactive immunophenotyping reagents for flow cytometry, and insufficient prior knowledge of cell type-defining markers.ResultsHere, we demonstrate the utility of single-cell RNA sequencing (scRNA-Seq) to characterize immune cells for which traditional experimental tools are limited. Specifically, we used scRNA-Seq to comprehensively define the cellular diversity of equine peripheral blood mononuclear cells (PBMC) from healthy horses across different breeds, ages, and sexes. We identified 30 cell type clusters partitioned into five major populations: monocytes/dendritic cells, B cells, CD3+PRF1+ lymphocytes, CD3+PRF1− lymphocytes, and basophils. Comparative analyses revealed many cell populations analogous to human PBMC, including transcriptionally heterogeneous monocytes and distinct dendritic cell subsets (cDC1, cDC2, plasmacytoid DC). Remarkably, we found that a majority of the equine peripheral B cell compartment is comprised of T-bet+ B cells, an immune cell subpopulation typically associated with chronic infection and inflammation in human and mouse.ConclusionsTaken together, our results demonstrate the potential of scRNA-Seq for cellular analyses in non-traditional model organisms and form the basis for an immune cell atlas of horse peripheral blood.

In-Depth Immune-Oncology Studies of the Tumor Microenvironment in a Humanized Melanoma Mouse Model.

The presence and interaction of immune cells in the tumor microenvironment is of significant importance and has a great impact on disease progression and response to therapy. Hence, their identification is of high interest for prognosis and treatment decisions. Besides detailed phenotypic analyses of immune, as well as tumor cells, spatial analyses is an important parameter in the complex interplay of neoplastic and immune cells—especially when moving into focus efforts to develop and validate new therapeutic strategies. Ex vivo analysis of tumor samples by immunohistochemistry staining methods conserves spatial information is restricted to single markers, while flow cytometry (disrupting tissue into single cell suspensions) provides access to markers in larger numbers. Nevertheless, this comes at the cost of scarifying morphological information regarding tissue localization and cell–cell contacts. Further detrimental effects incurred by, for example, tissue digestion include staining artifacts. Consequently, ongoing efforts are directed towards methods that preserve, completely or in part, spatial information, while increasing the number of markers that can potentially be interrogated to the level of conventional flow cytometric methods. Progression in multiplex immunohistochemistry in the last ten years overcame the limitation to 1–2 markers in classical staining methods using DAB with counter stains or even pure chemical staining methods. In this study, we compared the multiplex method Chipcytometry to flow cytometry and classical IHC-P using DAB and hematoxylin. Chipcytometry uses frozen or paraffin-embedded tissue sections stained with readily available commercial fluorophore-labeled antibodies in repetitive cycles of staining and bleaching. The iterative staining approach enables sequential analysis of a virtually unlimited number of markers on the same sample, thereby identifying immune cell subpopulations in the tumor microenvironment in the present study in a humanized mouse melanoma model.

Absolute quantification of tumor-infiltrating immune cells in high-grade glioma identifies prognostic and radiomics values.

To understand the tumor immune microenvironment precisely, it is important to secure the quantified data of tumor-infiltrating immune cells, since the immune cells are true working unit. We analyzed unit immune cell number per unit volume of core tumor tissue of high-grade gliomas (HGG) to correlate their immune microenvironment characteristics with clinical prognosis and radiomic signatures. The number of tumor-infiltrating immune cells from 64 HGG core tissue were analyzed using flow cytometry and standardized. After sorting out patient groups according to diverse immune characteristics, the groups were tested if they have any clinical prognostic relevance and specific radiomic signature relationships. Sparse partial least square with discriminant analysis using multimodal magnetic resonance images was employed for all radiomic classifications. The median number of CD45 + cells per one gram of HGG core tissue counted 865,770 cells which was equivalent to 8.0% of total cells including tumor cells. There was heterogeneity in the distribution of immune cell subpopulations among patients. Overall survival was significantly better in T cell-deficient group than T cell-enriched group (p = 0.019), and T8 dominant group than T4 dominant group (p = 0.023). The number of tumor-associated macrophages (TAM) and M2-TAM was significantly decreased in isocitrate dehydrogenase mutated HGG. Radiomic signature classification showed good performance in predicting immune phenotypes especially with features extracted from apparent diffusion coefficient maps. Absolute quantification of tumor-infiltrating immune cells confirmed the heterogeneity of immune microenvironment in HGG which harbors prognostic impact. This immune microenvironment could be predicted by radiomic signatures non-invasively.

Altered Spatial Composition of the Immune Cell Repertoire in Association to CD34+ Blasts in Myelodysplastic Syndromes and Secondary Acute Myeloid Leukemia.

Simple SummaryDespite a relationship between immune dysregulation and the course of myelodysplastic syndromes (MDS) has been discussed, a detailed understanding of this phenomenon is still missing. Therefore, multiplex analyses of bone marrow biopsies (BMB) from patients with MDS and secondary acute myeloid leukemia (sAML) were performed in order to determine the repertoire of lymphocyte subpopulations and their distance to CD34+ blasts. In MDS and sAML samples, the composition, quantity, and spatial proximity of immune cell subsets to CD34+ blasts were heterogeneous and correlated to the blast counts, but not to the genetics of the diseases, while in non-neoplastic BMB no CD8+ and FOXP3+ T cells and only single MUM1p+ B/plasma cells were detected in a distance of ≤10 μm to CD34+ hematopoietic progenitor cells (HPSC). We conclude that CD8+ and FOXP3+ T cells are not part of the immediate surrounding of CD34+ HPSC.Background: Myelodysplastic syndromes (MDS) are caused by a stem cell failure and often include a dysfunction of the immune system. However, the relationship between spatial immune cell distribution within the bone marrow (BM), in relation to genetic features and the course of disease has not been analyzed in detail. Methods: Histotopography of immune cell subpopulations and their spatial distribution to CD34+ hematopoietic cells was determined by multispectral imaging (MSI) in 147 BM biopsies (BMB) from patients with MDS, secondary acute myeloid leukemia (sAML), and controls. Results: In MDS and sAML samples, a high inter-tumoral immune cell heterogeneity in spatial proximity to CD34+ blasts was found that was independent of genetic alterations, but correlated to blast counts. In controls, no CD8+ and FOXP3+ T cells and only single MUM1p+ B/plasma cells were detected in an area of ≤10 μm to CD34+ HSPC. Conclusions: CD8+ and FOXP3+ T cells are regularly seen in the 10 μm area around CD34+ blasts in MDS/sAML regardless of the course of the disease but lack in the surrounding of CD34+ HSPC in control samples. In addition, the frequencies of immune cell subsets in MDS and sAML BMB differ when compared to control BMB providing novel insights in immune deregulation.

Single-cell profiling of the human decidual immune microenvironment in patients with recurrent pregnancy loss

Maintaining homeostasis of the decidual immune microenvironment at the maternal–fetal interface is essential for placentation and reproductive success. Although distinct decidual immune cell subpopulations have been identified under normal conditions, systematic understanding of the spectrum and heterogeneity of leukocytes under recurrent miscarriage in human deciduas remains unclear. To address this, we profiled the respective transcriptomes of 18,646 primary human decidual immune cells isolated from patients with recurrent pregnancy loss (RPL) and healthy controls at single-cell resolution. We discovered dramatic differential distributions of immune cell subsets in RPL patients compared with the normal decidual immune microenvironment. Furthermore, we found a subset of decidual natural killer (NK) cells that support embryo growth were diminished in proportion due to abnormal NK cell development in RPL patients. We also elucidated the altered cellular interactions between the decidual immune cell subsets in the microenvironment and those of the immune cells with stromal cells and extravillous trophoblast under disease state. These results provided deeper insights into the RPL decidual immune microenvironment disorder that are potentially applicable to improve the diagnosis and therapeutics of this disease.

Single-cell immune checkpoint landscape of PBMCs stimulated with Candida albicans

ABSTRACT Immune checkpoints play various important roles in tumour immunity, which usually contribute to T cells’ exhaustion, leading to immunosuppression in the tumour microenvironment. However, the roles of immune checkpoints in infectious diseases, especially fungal infection, remain elusive. Here, we reanalyzed a recent published single-cell RNA-sequencing (scRNA-seq) data of peripheral blood mononuclear cells (PBMCs) stimulated with Candida albicans (C. albicans), to explore the expression patterns of immune checkpoints after C. albicans bloodstream infection. We characterized the heterogeneous pathway activities among different immune cell subpopulations after C. albicans infection. The CTLA-4 pathway was up-regulated in stimulated CD4+ and CD8+ T cells, while the PD-1 pathway showed high activity in stimulated plasmacytoid dendritic cell (pDC) and monocytes. Importantly, we found that immunosuppressive checkpoints HAVCR2 and LAG3 were only expressed in stimulated NK and CD8+ T cells, respectively. Their viabilities were validated by flow cytometry. We also identified three overexpressed genes (ISG20, LY6E, ISG15) across all stimulated cells. Also, two monocyte-specific overexpressed genes (SNX10, IDO1) were screened out in this study. Together, these results supplemented the landscape of immune checkpoints in fungal infection, which may serve as potential therapeutic targets for C. albicans infection. Moreover, the genes with the most relevant for C. albicans infection were identified in this study.

Regulation and biological functions of the CX3CL1-CX3CR1 axis and its relevance in solid cancer: A mini-review

CX3CL1 is a transmembrane protein from which a soluble form can be generated by proteolytic shedding. Membranal and soluble forms of CX3CL1 exhibit different functions, although both bind to the CX3CR1 chemokine receptor. The CX3CL1-CX3CR1 axis mediates the adhesion of leukocytes and is also involved in cell survival and recruitment of immune cell subpopulations. The function of CX3CL1 is finely tuned by cytokines and transcription factors regulating its expression and post-translational modifications. On homeostasis, the CX3CL1-CX3CR1 axis participates in the removal of damaged neurons and neurogenesis, and it is also involved on several pathological contexts. The CX3CL1-CX3CR1 axis induces several cellular responses relevant to cancer such as proliferation, migration, invasion and apoptosis resistance. In this review, we address biological aspects of this molecular axis with important therapeutic potential, emphasizing its role in cancer, one of the most prevalent chronic diseases which significantly affect the quality of life and life expectancy of patients.

Infection and immune response to porcine hemagglutinating encephalomyelitis virus in grower pigs

Porcine hemagglutinating encephalomyelitis virus (PHEV) is the cause of acute outbreaks of vomiting and wasting disease and/or encephalomyelitis in neonatal pigs, with naïve herds particularly vulnerable to clinical episodes. PHEV infections in older pigs are generally considered to be subclinical, but are poorly characterized in the refereed literature. In this study, twelve 7-week-old pigs were oronasally inoculated with 0.5 mL (1:128 HA titer) PHEV (Mengeling strain) and then followed through 42 days post inoculation (dpi). Fecal and oral fluid specimens were collected daily to evaluate viral shedding. Serum samples were tested for viremia, isotype-specific antibody responses, cytokine, and chemokine responses. Peripheral blood mononuclear cells were isolated to evaluate phenotype changes in immune cell subpopulations. No clinical signs were observed in PHEV inoculated pigs, but virus was detected in oral fluid (1–28 dpi) and feces (1–10 dpi). No viremia was detected, but a significant IFN-α response was observed in serum at 3 dpi, followed by the detection of IgM (dpi 7), and IgA/IgG (dpi 10). Flow cytometry revealed a one-off increase in cytotoxic T cells at 21 dpi. This study demonstrated that exposure of grower pigs to PHEV results in subclinical infection characterized by active viral replication and shedding followed by an active humoral and cell-mediated immune response that attenuates the course of the infection and results in viral clearance.

Agile workflow for interactive analysis of mass cytometry data.

MotivationSingle-cell proteomics technologies, such as mass cytometry, have enabled characterization of cell-to-cell variation and cell populations at a single-cell resolution. These large amounts of data, require dedicated, interactive tools for translating the data into knowledge.ResultsWe present a comprehensive, interactive method called Cyto to streamline analysis of large-scale cytometry data. Cyto is a workflow-based open-source solution that automates the use of state-of-the-art single-cell analysis methods with interactive visualization. We show the utility of Cyto by applying it to mass cytometry data from peripheral blood and high-grade serous ovarian cancer (HGSOC) samples. Our results show that Cyto is able to reliably capture the immune cell sub-populations from peripheral blood and cellular compositions of unique immune- and cancer cell subpopulations in HGSOC tumor and ascites samples.Availabilityand implementationThe method is available as a Docker container at https://hub.docker.com/r/anduril/cyto and the user guide and source code are available at https://bitbucket.org/anduril-dev/cyto. Supplementary information Supplementary data are available at Bioinformatics online.

LDOC1 as Negative Prognostic Marker for Vulvar Cancer Patients.

So far, studies about targeted therapies and predictive biomarkers for vulva carcinomas are rare. The leucine zipper downregulated in cancer 1 gene (LDOC1) has been identified in various carcinomas as a tumor-relevant protein influencing patients’ survival and prognosis. Due to the lack of information about LDOC1 and its exact functionality, this study focuses on the expression of LDOC1 in vulvar carcinoma cells and its surrounding immune cells as well as its correlation to clinicopathological characteristics and prognosis. Additionally, a possible regulation of LDOC1 in vulvar cancer cell lines via the NF-κB signaling pathway was analyzed. Vulvar carcinoma sections of 157 patients were immunohistochemically stained and examined regarding LDOC1 expression by using the immunoreactive score (IRS). To characterize LDOC1-positively stained immune cell subpopulations, immunofluorescence double staining was performed. The effect of the NF-κB inhibitor C-DIM 12 (3,3′-[(4-chlorophenyl)methylene]bis[1 H-indole]) on vulvar cancer cell lines A431 and SW 954 was measured according to MTT and BrdU assays. Baseline expression levels of LDOC1 in the vulvar cancer cell lines A431 and SW 954 was analyzed by real-time PCR. LDOC1 was expressed by about 90% of the cancer cells in the cytoplasm and about half of the cells in the nucleus. Cytoplasmatic expression of LDOC1 was associated with decreased ten-year overall survival of the patient, whereas nuclear staining showed a negative association with disease-free survival. Infiltrating immune cells were mainly macrophages followed by regulatory T cells. Incubation with C-DIM 12 decreased the cell viability and proliferation of vulvar cancer cell line A431, but not of cell line SW 954. LDOC1 expression on mRNA level was twice as high in the cell line A431 compared to the cell line SW 954. Overexpression of LDOC1 was associated with unfavorable overall and disease-free survival. Tumor growth could be inhibited by C-DIM 12 in vitro if the expressed LDOC1 level was high enough.

Anacardic 6-pentadecyl salicylic acid induces apoptosis in breast cancer tumor cells, immunostimulation in the host and decreases blood toxic effects of taxol in an animal model

Many antineoplastic agents induce myelosuppression and leukopenia as secondary effects in patients. The development of anticancer agents that simultaneously provoke antitumor immune response represents an important therapeutic advance. The administration of 6-pentadecyl salicylic acid (6SA) contributes to the antitumor immunity using 4T1 breast cancer cells in Balb/c female mice, with Taxol as a positive control and in cotreatment with 6SA (6SA + Taxol; CoT).Our results show that 6SA reduces tumor volume and size by inducing caspase-8-mediated apoptosis without reducing tumor infiltrated lymphocytes. Also, 6SA reduced lung metastasis and increased the proportion of immune cells in blood, lymph nodes and bone marrow; more evidently, in the proportion of tumor-infiltrated natural killer (NK) cells and cytotoxic T lymphocytes. Taxol reduces helper and cytotoxic lymphocytes causing systemic immunosuppression and myelosuppression in bone marrow, whereas 6SA does not decrease any immune cell subpopulations in circulating blood and lymph nodes. More importantly, the CoT decreased the Taxol-induced cytotoxicity in circulating T cells and bone marrow. Treatment with 6SA increases the secretion of IL-2, IL-12, GM-CSF, TNF-α and IFN-γ and significantly reduces IL-10 and IL-17 secretion, suggesting that the reduction of regulatory T cells and tumor-associated macrophages contribute to the host control of tumor development.Finally, 6SA has an effective antineoplastic activity against breast cancer cells in an immunocompetent animal, reduces the myelosuppression and leukopenia that Taxol produces, improves the antitumoral immunological microenvironment and increases the overall survival of the animals improving the quality of life of patients with cancer.

Identification of distinct immune activation profiles in adult humans

Latent infectious agents, microbial translocation, some metabolites and immune cell subpopulations, as well as senescence modulate the level and quality of activation of our immune system. Here, we tested whether various in vivo immune activation profiles may be distinguished in a general population. We measured 43 markers of immune activation by 8-color flow cytometry and ELISA in 150 adults, and performed a double hierarchical clustering of biomarkers and volunteers. We identified five different immune activation profiles. Profile 1 had a high proportion of naïve T cells. By contrast, Profiles 2 and 3 had an elevated percentage of terminally differentiated and of senescent CD4+ T cells and CD8+ T cells, respectively. The fourth profile was characterized by NK cell activation, and the last profile, Profile 5, by a high proportion of monocytes. In search for etiologic factors that could determine these profiles, we observed a high frequency of naïve Treg cells in Profile 1, contrasting with a tendency to a low percentage of Treg cells in Profiles 2 and 3. Moreover, Profile 5 tended to have a high level of 16s ribosomal DNA, a direct marker of microbial translocation. These data are compatible with a model in which specific causes, as the frequency of Treg or the level of microbial translocation, shape specific profiles of immune activation. It will be of interest to analyze whether some of these profiles drive preferentially some morbidities known to be fueled by immune activation, as insulin resistance, atherothrombosis or liver steatosis.

Single-Cell Immune Profiling in Coronary Artery Disease: The Role of State-of-the-Art Immunophenotyping With Mass Cytometry in the Diagnosis of Atherosclerosis.

Coronary artery disease remains the leading cause of death globally and is a major burden to every health system in the world. There have been significant improvements in risk modification, treatments, and mortality; however, our ability to detect asymptomatic disease for early intervention remains limited. Recent discoveries regarding the inflammatory nature of atherosclerosis have prompted investigation into new methods of diagnosis and treatment of coronary artery disease. This article reviews some of the highlights of the important developments in cardioimmunology and summarizes the clinical evidence linking the immune system and atherosclerosis. It provides an overview of the major serological biomarkers that have been associated with atherosclerosis, noting the limitations of these markers attributable to low specificity, and then contrasts these serological markers with the circulating immune cell subtypes that have been found to be altered in coronary artery disease. This review then outlines the technique of mass cytometry and its ability to provide high‐dimensional single‐cell data and explores how this high‐resolution quantification of specific immune cell subpopulations may assist in the diagnosis of early atherosclerosis in combination with other complimentary techniques such as single‐cell RNA sequencing. We propose that this improved specificity has the potential to transform the detection of coronary artery disease in its early phases, facilitating targeted preventative approaches in the precision medicine era.

TGF-β2 is a Prognostic Biomarker Correlated with Immune Cell Infiltration in Colorectal Cancer: A STROBE-compliant article.

Transforming growth factor-beta (TGF-β2) is an important cytokine regulating immune cell function. However, whether TGF-β2 controls the invasion of colorectal cancer (CRC) by immune cells is unknown. Therefore, we evaluated the expression of TGF-β2 using multiple databases and determined the relationship between TGF-β2 expression and tumor immune infiltration defined by a set of genetic markers. The analysis demonstrated that the expression of TGF-β2 is closely related to the outcome of many cancers, and this correlation was particularly strong in CRC. In addition, the increased expression of TGF-β2 was significantly associated with the expression of various markers of specific immune cell subpopulations, and overexpression of TGF-β2 was closely related to the prognosis of colon cancer patients. Moreover, TGF-β2 was related to the prognosis and infiltration of the tumor by immune cells in CRC patients. The obtained results indicate that TGF-β2 is a critical factor regulating the recruitment of immune cells and controls their infiltration into colorectal tumors. Thus, high expression of TGF-β2 not only facilitates the prognosis in CRC patients, but also may provide a new target for the treatment of CRC.

Defibrotide for the Treatment of Endotheliitis Complicating Sars-Cov-2 Infection: Rationale and Ongoing Studies As Part of the International Defacovid Study Group

Coronavirus disease 2019 (COVID-19) is a heterogeneous clinical condition caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The initial stage of infection occurs at the time of respiratory viral invasion through angiotensin-converting enzyme 2 (ACE2) receptors in alveolar pneumocytes and macrophages. In the second stage of established pulmonary disease without and with hypoxia, viral multiplication and localized inflammation occurs. The third stage COVID-19 is the most severe, characterized by systemic hyperinflammation, acute respiratory distress syndrome (ARDS) and generalized microangiopathy, with significantly elevated biomarkers including IL-2, IL-6, IL-7, granulocyte-colony stimulating factor, macrophage inflammatory protein 1α, tumor necrosis factor-α, C-reactive protein, ferritin, D-dimer, von Willebrand factor (vWF) and thrombomodulin (TM). Evidence suggests that endothelial damage and activation play a key role in the pathobiology of the disease, and thus protecting endothelium and reversing endotheliitis may be a key therapeutic goal (Teuwen et al,Nat Rev Immunol2020).Defibrotide (DF), a complex mixture of poly-deoxyribonucleotides extracted from porcine mucosa, modulates expression of thrombogenic and inflammatory mediators produced in response to endothelial cell injury and/or leukocyte activation. DF increases tissue plasminogen activator (t-PA) and TM expression, thereby enhancing the enzymatic activity of plasmin to hydrolyze fibrin clot, while also decreasing vWF and plasminogen activator inhibitor-1 (PAI-1). Platelet adhesion is inhibited via increases in nitric oxide (NO), prostaglandin I2 (PGI2), and prostaglandin E2 (PGE2) (Falanga A et al,Leukemia 2003). Anti-inflammatory properties via inhibition of p38 MAPK pathway have been demonstrated with DF, attenuating the release of inflammatory mediators including IL-6, thromboxane A2, leukotriene B4, tumor necrosis factor-alpha, and reactive oxygen species (Mitsiades et al,Clin Cancer Res2009). Additionally, DF inhibits both the expression and activity of heparanase, which in turn modulates heparan sulfate, a vital moiety for viral infection (Richardson et al,Blood Adv2018; Koganti et al,Cell Mol Life Sci2020).DF also modulates endothelial cell injury in murine models by down-regulating the expression of endothelial cell adhesion molecules such as E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), that are critical to leukocyte adhesion and migration, and by increasing the endothelial cell release of anti-inflammatory cytokines, conferring survival benefit (Garcia-Bernal et al,J Cell Mol Med2020). DF is approved for the treatment of severe veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS) complicating stem cell transplantation and has demonstrated efficacy and safety in critically ill patients with multi-organ failure, underpinned by vasculopathy, endotheliitis, immune activation and hemorrhagic risk (Richardson et al,Blood2016). This multi-target and pleiotropic approach makes DF an attractive candidate for the treatment of advanced stage COVID 19 and the systemic endothelial complications underpinning both its pathobiology and ensuing mortality (Figure 1).Various clinical studies in Spain, Italy, Ireland, the UK and USA are underway, with the larger randomized placebo-controlled prospective phase 2 study in Spain (clinicaltrials.gov: NCT04348383) leading in accrual with promising early results, demonstrating safety to date with potential efficacy, as reflected by favorable outcome in a subset of intubated patients as part of an ongoing multi-center trial. Other studies within the group have focused on safety and particularly the therapeutic role of DF in advanced disease, including those patients requiring extracorporeal membrane oxygenation (ECMO) and full dose systemic anticoagulation. These trials include common in-depth biological correlatives, assessing biomarkers including heparanase, inflammatory cytokines, immune cell sub-populations, vWF and soluble TM, as well as other novel markers and pharmacokinetics. In aggregate, these studies will hopefully help demonstrate the effects of DF on the proposed targets in COVID-19 and successfully correlate these effects with improved clinical outcome.DisclosuresMoraleda:Jazz Pharmaceuticals:Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding;Novartis, Sandoz, Takeda:Honoraria;Gilead:Honoraria, Other: Travel expenses.Carlo-Stella:Servier, Novartis, Genenta Science srl, ADC Therapeutics, F. Hoffmann-La Roche, Karyopharm, Jazz Pharmaceuticals:Membership on an entity’s Board of Directors or advisory committees;Bristol-Myers Squibb, Merck Sharp & Dohme, Janssen Oncology, AstraZeneca:Honoraria;Boehringer Ingelheim and Sanofi:Consultancy;ADC Therapeutics and Rhizen Pharmaceuticals:Research Funding.Jara Rubio:Edwards Lifesciences, Getinge:Honoraria.Diaz-Ricart:Jazz Pharmaceuticals:Honoraria, Research Funding;German Jose Carreras Leukaemia Foundation:Research Funding.Carreras:Jazz Pharmaceuticals:Research Funding, Speakers Bureau;German Jose´ Carreras Leukaemia Foundation:Research Funding.Mo:Celgene/BMS:Membership on an entity’s Board of Directors or advisory committees.Cooke:Jazz Pharmaceuticals:Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau.Soiffer:Be the Match/ National Marrow Donor Program:Membership on an entity’s Board of Directors or advisory committees;alexion:Consultancy;Celgene:Membership on an entity’s Board of Directors or advisory committees;Juno:Membership on an entity’s Board of Directors or advisory committees;Novartis:Consultancy;VOR Biopharma:Consultancy;Mana Therapeutics:Consultancy;Precision Bioscience:Consultancy;Cugene:Consultancy;Rheos Therapeutics:Consultancy;Gilead:Consultancy;Kiadis:Membership on an entity’s Board of Directors or advisory committees.Baron:Genetech:Consultancy;Merck:Consultancy.Pagliuca:Jazz Pharmaceuticals:Membership on an entity’s Board of Directors or advisory committees.Richardson:Celgene/BMS, Oncopeptides, Takeda, Karyopharm:Research Funding.OffLabel Disclosure:Defibrotide used for the treatment of endotheliitis complicating SARS-Cov-2 infection

Sars-Cov-2 Spike Protein Induces Cellular Changes in Primitive and Mature Hematopoietic Cells

The devastating effects of SARS-CoV-2 infection (which causes COVID-19) highlight the need for a deeper understanding of the virus, host response, and the tissues affected by infection, including the hematopoietic stem cells (HSC), hematopoietic progenitors (HPC), and more mature blood cells including immune cells. While other coronavirus infections have been associated with hematopoietic or immune cell complications (Chu et al., J Infect Dis 2016), the effects of SARS-CoV-2 infection on HSC/HPC and mature immune cells are not yet understood. We performed studies to examine the potential susceptibility of HSC/HPC subpopulations and immune cells to infection and functional studies to explore if exposure to SARS-CoV-2 proteins impacts these cells. The most well-studied receptor for SARS-CoV-2 is ACE2 (Hoffmann et al., Cell 2020), a cell surface protein to which SARS-CoV-2 spike protein (S protein) binds to facilitate viral entry to host cells. We explored expression of ACE2 in primitive and mature blood cells. We demonstrated using RT-qPCR as well as western blotting that ACE2 is expressed at both the mRNA and protein levels in pooled low-density peripheral blood cells (PB) from healthy donors (n=5); pooled CD34+ cells from cord blood (CB) enriched for HSC/HPC (n=7); lineage+ (lin+) low density CB cells (n=7), and high density polymorphonuclear leukocytes (PMN) from CB (n=3). We then determined ACE2 expression on specific subpopulations of HSC/HPC and immune cells. Flow cytometry analysis (FACS) demonstrated that ACE2 is expressed on the cell surface of small subpopulations of PB immune cells, including 1-2% of T-cells, 2-4% of B-cells, and <1% of NK cells and monocytes. However, larger populations of HSC/HPC exhibited ACE2 cell surface expression. The percent ACE2+ HSC, multipotent progenitor cells (MPP), and multipotent lymphoid progenitor cells (MLP) were respectively 15-60%, 5-50%, and 5-15% of cells (n=5). We next determined if exposure to viral S protein affects these cell populations. We first tested if SARS-CoV-2 S protein induces responses in HSC/HPC. We isolated CD34+ cells from low density CB and expanded them in the presence of serum and growth factors (100ng/mL TPO, FLT3L, and SCF) in the presence or absence of recombinant full-length S protein. After 7 days expansion, cells were analyzed for immunophenotype using FACS or were plated in semi-solid methylcellulose in an HPC colony forming unit (CFU) assay and grown for 12 days before scoring for functional CFU-Granulocyte/Erythroid/Macrophage/Megakaryocyte (CFU-GEMM) and CFU-Granulocyte/Macrophage (CFU-GM) progenitor cells. CB CD34+ cells exhibit significantly less expansion when exposed to SARS-CoV-2 S protein, showing 33% less expansion of CD34+ cells. The most significant delay in expansion of phenotyped cells is evident in granulocyte-monocyte progenitors (GMP, 38% less expansion), with a more modest 15-30% decrease in expansion for common myeloid progenitors/megakaryocyte-erythroid progenitors (CMP/MEP), HSC, MPP, and MLP (n=4). S protein lessens functional HPC colony forming cell expansion compared to control, with 12-fold and 2.1-fold expansions compared to 30.8-fold and 6-fold expansions respectively for CFU-GM and CFU-GEMM. These data indicate that exposure to SARS-CoV-2 S protein impacts hematopoiesis and myeloid differentiation in vitro. We next tested effects of S protein exposure on PB by isolating low density blood cells from healthy donors and culturing them ex vivo for different time periods in the presence or absence of recombinant full-length S protein. After 2hrs incubation, we observed by FACS a modest but consistent 1.2-fold increase in CD14 positive monocytes in cells exposed to S protein compared vehicle control (n=4). After 18hrs incubation with the S protein, the PB monocytes exhibited consistent changes in morphology, becoming smaller and more granular, determined by changes in forward scatter and side scatter by FACS (n=5). This suggests that exposure to SARS-CoV-2 protein may have a physiologic impact on PB cells ex vivo, despite their low levels of ACE2 expression. Our data thus has important implications for hematopoiesis and immune response in COVID-19 patients. It demonstrates the need to determine whether CB potentially exposed to SARS-CoV-2 should be banked, as the effects we observed may affect the efficacy of CB transplantations.DisclosuresNo relevant conflicts of interest to declare.

Optimized in vitro isolation of different subpopulation of immune cells from peripheral blood and comparative techniques for generation of monocyte-derived macrophages in small ruminants

Peripheral blood from healthy sheep (n = 3) and goats (n = 3) were employed to establish an efficient method for simultaneous isolation of peripheral blood mononuclear cells (PBMCs) and neutrophils and to standardize protocols for monocyte purification and generation of monocyte-derived macrophages (MDMs). In both species, a significantly enriched population of PBMCs, with higher purity and number of cells determined by flow cytometry, was achieved when processing through a density gradient a mixture of buffy-coat and red blood cell layer (RBC) in comparison to the use of just the buffy-coat (p < 0.05). Neutrophils could be subsequently isolated from the layer, located underneath PBMCs fraction with significant higher purity rates, higher than 85 % determined by flow cytometry, than those obtained with protocols without density gradients (< 60 %) (p < 0.05). This technique would allow the isolation of both cell populations from the same sample of blood. A pure cell population of monocytes, CD14+ cells, was purified from PBMCs when using immunomagnetic columns, which allow for 17 % (nº monocytes/nº PBMCs) of yield and high percentages of expression of CD14+ (88 %), MHC-II+ (91.5 %) and CD11b+ (94 %) established by flow cytometry. On the other hand, the classical and non-expensive purification of monocytes from PBMCs based on the adherence capacity of the former, allowed significantly lower yield of monocytes (4.6 %), with percentages of surface markers expression that dropped to 35 %, 65 % and 55 %, respectively (p < 0.001), suggesting the isolation of a mixed population of cells. The addition of GM-CSF to the culture, at concentration from 25 to 125 ng/mL, enhanced proportionally the number of MDMs generated compared to the absence of supplementation or the use of autologous serum from 5% to 20 %. However, purification of monocytes through the adherence method achieved higher yields of MDMs than those isolated through immunomagnetic columns in both species (p < 0.001). Under the conditions of this study, the use of centrifugation in density gradients allow for the simultaneous purification of PBMCs and neutrophils, with high purity of both populations, from the same sample of blood. The isolation of monocytes could be subsequently achieved through two different methods, i.e. based on immunomagnetic columns or adherence. The preference between both methods would depend on the necessities of the experiment, the initial sample with high purity of monocytes or a final population of MDMs required.