Peripheral Blood Of Healthy Individuals Research Articles

Differences in the direct effects of various type 2 cytokines on functions of blood eosinophils from healthy subjects.

Eosinophil inflammation often persists in the airways of severe asthmatics, even under treatment with high-dose inhaled corticosteroids. Biologics for various type 2 cytokines have been recently developed for corticosteroid-resistant, eosinophil-dominant, severe asthma. However, it is unclear whether these biologics act directly on eosinophils. In this study, we examined whether various type 2 cytokines targeted by biologics can directly modify the functions of eosinophils obtained from the peripheral blood of healthy individuals. Peripheral eosinophils of healthy subjects were purified by conventional negative-depletion methods using anti-CD16 beads to avoid the priming effect (i.e., stimulation in vitro) to the maximum extent possible. Eosinophils were stimulated with interleukin (IL)-4, IL-5, IL-13, or thymic stromal lymphopoietin (TSLP), and eosinophil adhesiveness to recombinant human-intercellular adhesion molecule (ICAM)-1 was evaluated by eosinophil peroxidase assays. The effect of these cytokines on eosinophil superoxide anion (O2 -) generation was evaluated by the superoxide dismutase-inhibitable reduction of cytochrome C. To determine whether eosinophil degranulation was induced, the concentration of eosinophil-derived neurotoxin (EDN) in the supernatant was measured using enzyme-linked immuno sorbent assay. As reported previously, at 100 pM, IL-5 increased eosinophil adhesiveness to ICAM-1, O2 - generation, and EDN release. Conversely, at concentrations up to 10 nM, IL-4, IL-13, and TSLP did not induce eosinophil adhesiveness, O2 - generation, or EDN release. Type 2 cytokines other than IL-5 do not directly affect the functions of eosinophils from healthy individuals when used at clinical concentrations. These findings suggest that eosinophils play little, or no, direct role in the effects of anti-IL-4 receptor α or anti-TSLP antibody on severe asthma.

NECA alleviates inflammatory responses in diabetic retinopathy through dendritic cell toll-like receptor signaling pathway.

This study examined the impact of 5'-(N- ethylcarboxamido)adenosine (NECA) in the peripheral blood of healthy individuals, those with diabetes mellitus, diabetic retinopathy (DR), and C57BL/6 mice, both in vivo and in vitro. Enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FCM) were used to evaluate the effects of NECA on dendritic cells (DCs) and mouse bone marrow-derived dendritic cells (BMDCs) and the effects of NECA-treated DCs on Treg and Th17 cells. The effect of NECA on the Toll-like receptor (TLR) pathway in DCs was evaluated using polymerase chain reaction (PCR) and western blotting (WB). FCM and ELISA showed that NECA inhibited the expression of surface markers of DCs and BMDCs, increased anti-inflammatory cytokines and decreased proinflammatory cytokines. PCR and WB showed that NCEA decreased mRNA transcription and protein expression in the TLR-4-MyD88-NF-kβ pathway in DCs and BMDCs. The DR severity in streptozocin (STZ) induced diabetic mice was alleviated. NECA-treated DCs and BMDCs were co-cultivated with CD4+T cells, resulting in modulation of Treg and Th17 differentiation, along with cytokine secretion alterations. NECA could impair DCs' ability to present antigens and mitigate the inflammatory response, thereby alleviating the severity of DR.

Abstract LB185: High resolution telomere measurements in human cancer and aging using long-read nanopore sequencing

Abstract Telomere length is an important biomarker of cellular replicative potential and aging, but existing measurement methods are limited in resolution and accuracy. During human carcinogenesis, telomeres shorten profoundly in the earliest stages that precede tumor invasion. Telomere shortening can either halt cancer progression by inducing senescence and cell death or can promote cancer development by destabilizing chromosomes and generating chromosomal rearrangements. Stabilization of telomeres, typically by the upregulation of telomerase, is required for progression of nearly all human cancers. Here, we deploy digital telomere measurement by nanopore sequencing to understand how distributions of human telomere length change during aging and following carcinogenesis. We measure telomere attrition and de novo elongation with unprecedented resolution in genetically defined populations of human stem cells, in cells from the peripheral blood of healthy individuals and patients with genetic defects in telomere maintenance, cancer patients, and cancer cell lines. We find that human aging is accompanied by a progressive loss of long telomeres and an accumulation of shorter telomeres in white blood cells. In patients with inherited defects in telomere maintenance, the accumulation of short telomeres is more pronounced and correlates with phenotypic severity. We apply machine learning to train a binary classification model that distinguishes healthy individuals from those with telomere biology disorders. Next, we investigated the telomere lengths of a cohort of twenty colorectal tumors with patient-matched healthy colonic epithelia and found that two-thirds of colorectal tumors have telomere length distributions significantly shorter than the surrounding normal tissue. Finally, we explore the utility of telomere length distributions in cancer cell lines as clinically actionable disease biomarkers. This sequencing and bioinformatic pipeline will advance our understanding of telomere maintenance mechanisms and the use of telomere length as a clinical biomarker of aging and disease. Citation Format: Santiago Sanchez, Jessica Gu, Anudeep Golla, William Shomali, Annika Martin, Dirk Hockemeyer, Sharon Savage, Steven Artandi. High resolution telomere measurements in human cancer and aging using long-read nanopore sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB185.

Abstract 5228: Engineering CD123-targeting vδ1-enriched γδT cells with enhanced preclinical efficacy and safety profile

Abstract Acute myeloid leukemia (AML) is an aggressive hematological malignancy that, despite advances in treatment, has an overall 5-year survival rate of less than 30%. There is an urgent unmet need for new and effective therapies for AML. Chimeric antigen receptor (CAR)-modified cell therapies are emerging as promising options. However, the selection of suitable antigens is challenging, due to the heterogeneous expression of potential antigen targets in AML. Most antigens, such as CD123, are expressed on both leukemic cells and normal hematopoietic stem and progenitor cells. In addition, conventional autologous or allogeneic T cell therapies present many limitations, such as cytokine release syndrome (CRS) and graft-vs-host disease (GvHD). We therefore evaluated CD123-targeting CAR designs in vδ1-enriched γδT cells, an innate immune cell platform that is inherently non-MHC restricted, has low risks of CRS and GvHD, and is capable of discriminating between malignant and healthy cells in preclinical models. We engineered vδ1-enriched γδT cells from peripheral blood of healthy individuals to express various CD123-targeting CAR constructs. The CD123-targeting CAR-engineered vδ1-enriched γδT cells expressed high levels of natural cytotoxicity receptors, suggesting capability to target innate receptor ligands on AML cell surface. CD123-targeting CAR-expressing vδ1-enriched γδT cells demonstrated enhanced cytotoxicity in vitro against multiple AML cell lines, in both short-term co-culture and repeated antigen stimulation assays. CD123-targeting CAR-expressing vδ1-enriched γδT cells also showed enhanced efficacy and survival in vivo against mouse models of human AML. Interestingly, attenuation of CAR activity via novel designs preserved the inherent selectivity of vδ1-enriched γδT cells against AML cells relative to normal hematopoietic stem or progenitor cells, which also express CD123. Together, our results demonstrated through rational CAR designing and engineering in vδ1-enriched γδT cells, an allogeneic cell therapy targeting CD123 can be generated with selective cytotoxicity against AML cells while sparing normal hematopoietic stem cells. Such product designs have the potential to be a transformative therapeutic option for a broad population of AML patients. Citation Format: Mei Rosa Ng, Sarah L. Hayward, Di Zhang, Gary Gu, Bri Paulhus, Donjeta Gjuka, Collin Crowley, Sophie McLaughlin, Anya Lublinsky, Angel Mathew, Evans Berreondo Giron, Doanh Mai, Madisen Lee, Megha Wal, Rebecca Alade, Andre Simoes, Robin Lytle, James Gavin, Shira Cramer, Zhong-hua Yan, Taylor Hickman, Rashmi Choudhary, Cori Abikoff, Yana Wang, Istvan Kovacs, Nandhu M. Sobhana, Lan Cao. Engineering CD123-targeting vδ1-enriched γδT cells with enhanced preclinical efficacy and safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5228.

Rituximab may affect T lymphocyte subsets balance in primary membranous nephropathy

BackgroundThe aim of this study was to investigate the effects and significance of rituximab (RTX) on the levels of T lymphocyte subsets in patients diagnosed with primary membranous nephropathy (PMN).MethodsA total of 58 PMN patients and 25 healthy donors were chosen as the subjects. Among the PMN patients, 40 individuals received RTX treatment and completed at least 6 months of follow-up. All subjects underwent flow cytometry analysis to determine the peripheral blood lymphocyte subsets. The changes in anti-PLA2R antibody titers and 24-hour urinary protein levels were evaluated by ELISA and Biuret method before and after treatment.Results(1) The PMN group exhibited a significantly greater percentage of peripheral blood CD3−CD19+ B cells than the healthy group, which is consistent with the findings of previous reports. Additionally, compared with those in the peripheral blood of healthy individuals, the numbers of CD4+ central memory T cells, CD4+ effector memory T cells, CD4+/CD8+, and CD4+CD25+ T cells in the PMN peripheral blood were markedly greater. However, the number of peripheral blood Treg cells was reduced in the PMN group. (2) After 6 months of RTX treatment, PMN patients exhibited significant decreases in anti-PLA2R antibody titers, 24-hour urinary protein levels, and peripheral blood CD3−CD19+ B cells. Importantly, RTX administration decreased CD4+CD25+ T cells and CD4+/CD8+ in the peripheral blood of PMN patients and improved Treg cell levels. (3) RTX treatment induced alterations in the CD4+ T lymphocyte subsets in PMN patients, which did not correlate with B lymphocyte counts or anti-PLA2R antibody titers.ConclusionsRTX treatment might have a beneficial impact on cellular immunity by effectively restoring the balance of CD4+ T lymphocyte subsets in PMN patients, which is beyond its effects on B cells and antibody production.Trial registrationThe research was registered at the First Affiliated Hospital of Soochow University. Registration Number: MR-32-23-016211. Registration Date: May 31, 2023.

Reactive Oxygen Species Modulate Th17/Treg Balance in Chlamydia psittaci Pneumonia via NLRP3/IL-1β/Caspase-1 Pathway Differentiation.

Chlamydia psittaci pneumonia (CPP) is a lung disease caused by the infection with the Chla-mydia psittaci bacterium, which can lead to severe acute respiratory distress syndrome and systemic symptoms. This study explored the specific mechanisms underlying the impact of reactive oxygen species (ROS) on the Th17/Treg balance in CPP. The levels of ROS and the differentiation ratio of Th17/Treg in the peripheral blood of healthy individuals and CPP patients were measured using ELISA and flow cytometry, respectively. The association between the ROS levels and Th17/Treg was assessed using Pearson correlation analysis. The ROS levels and the Th17/Treg ratio were measured in CD4+ T cells following H2O2 treatment and NLRP3 inhibition. The effects of H2O2 treatment and NLRP3 inhibition on the NLRP3/IL-1β/caspase-1 pathway were observed using immunoblotting. Compared to the healthy group, the CPP group exhibited increased levels of ROS in the peripheral blood, an elevated ratio of Th17 differentiation, and a decreased ratio of Treg differentiation. ROS levels were positively correlated with the Th17 cell proportion but negatively correlated with the Treg cell proportion. The ROS levels and NLRP3/IL-1β/caspase-1 expression were up-regulated in CD4+ T cells after H2O2 treatment. Furthermore, there was an increase in Th17 differentiation and a decrease in Treg differentiation. Conversely, the NLRP3/IL-1β/caspase-1 pathway inhibition reversed the effects of H2O2 treatment, with no significant change in the ROS levels. ROS regulates the Th17/Treg balance in CPP, possibly through the NLRP3/IL-1β/caspase-1 pathway. This study provides a new perspective on the development of immunotherapy for CPP.

Neutrophil extracellular traps are involved in the occurrence of interstitial lung disease in a murine experimental autoimmune myositis model.

The excessive formation of neutrophil extracellular traps (NETs) has been demonstrated to be a pathogenic mechanism of idiopathic inflammatory myopathy (IIM)-associated interstitial lung disease (ILD). This study aimed to answer whether an experimental autoimmune myositis (EAM) model can be used to study IIM-ILD and whether NETs participate in the development of EAM-ILD. An EAM mouse model was established using skeletal muscle homogenate and pertussis toxin (PTX). The relationship between NETs and the ILD phenotype was determined via histopathological analysis. As NETs markers, serum cell-free DNA (cfDNA) and serum citrullinated histone 3 (Cit-H3)-DNA were tested. The healthy mouse was injected with PTX intraperitoneally to determine whether PTX intervention could induce NETs formation in vivo. Neutrophils isolated from the peripheral blood of healthy individuals were given different interventions to determine whether PTX and skeletal muscle homogenate can induce neutrophils to form NETs in vitro. EAM-ILD had three pathological phenotypes similar to IIM-ILD. Cit-H3, neutrophil myeloperoxidase, and neutrophil elastase were overexpressed in the lungs of EAM model mice. The serum cfDNA level and Cit-H3-DNA complex level were significantly increased in EAM model mice. Serum cfDNA levels were increased significantly in vivo intervention with PTX in mice. Both PTX and skeletal muscle homogenate-induced neutrophils to form NETs in vitro. EAM-ILD pathological phenotypes are similar to IIM-ILD, and NETs are involved in the development of ILD in a murine model of EAM. Thus, the EAM mouse model can be used as an ideal model targeting NETs to prevent and treat IIM-ILD.

MICROVESICLE SURFACE PROTEIN DETECTION AS A POTENTIAL BIOMARKER IN COLORECTAL CANCER

Abstract Introduction Colorectal cancer (CRC) is the third leading cause of death from cancer in the world; early detection is a key factor for patient´s prognosis. Microvesicles (MVs) are subcellular structures delimited by lipid bilayer intimately involved in physio-pathological processes such as cancer. Our objective is the characterization and detection of surface proteins in plasma derived MVs, as well as their usefulness as a tool in the detection of CRC. Methods Circulating MVs in peripheral blood of healthy individuals, with adenomas, high grade adenoma or carcinoma were isolated and characterized. MVs CD9, CD147 and EpCaM MVs surface proteins quantification was performed using an innovative lateral flow immunoassay (LFIA) developed in our laboratory. Results The characterization of the plasma MVs confirmed our previous observations, presenting a lower concentration of MVs in the groups of high-grade adenoma and mainly CRC. However, the carcinoma group presents a higher amount of protein per MV suggesting a greater MV´s complexity in this group. The analysis of surface antigens by LFIA show an unexpectedly significant reduction of these proteins in the carcinoma group. Conclusions The detection of antigens on the surface of MVs by LFIA may be able to discriminate patients with CRC and could emerge as an alternative test that complements CRC screening programs.

CD3<sup>+</sup>CD294<sup>+</sup>T cells of the type 2 immune response: their role in allergic inflammation

T lymphocytes type 2 immune response protect against helminths and toxins, and also contribute to the development of allergic inflammation. One of the most specific T cell surface markers T lymphocytes 2 is the CRTH2 molecule (CD294), which is an activating receptor for prostaglandin D2. The CD3+CD294+population is negligible in the peripheral blood of healthy individuals; an increase occurs in patients with allergic diseases and an autoimmune nature of the response. The aim of the study was to study the quantitative and functional characteristics Т lymphocytes type 2 immune response in patients with rhinoconjunctival symptoms (n = 248) and drug hypersensitivity (n = 184). In 68 patients with an elevated and extremely high number of CD3+CD294+cells, a detailed phenotype of this population was characterized by flow cytometry and the functional activity of the studied cell population in relation to the production of interleukin 4 and interferon γ was studied using enzyme immunoassay. The control group consisted of 34 volunteers. The relative number of CD3+CD294+cells was significantly higher in the group of patients with drug hypersensitivity – 1.6% and rhinoconjunctival symptoms 1.2% compared to the control group – 1.0%. Elevated (1.6-3.6%) and extremely high (>3.6%) CD3+CD294+cell number were significantly more frequently detected in patients with drug hypersensitivity. In both groups, an increase in the number of CD3+CD294+cells were observed with severe damage to the skin. The phenotype of the population T lymphocytes type 2 CD45RA-CD3+CD294+CD2+CD5+CD7+CD27+CD28+CD57-CCR7-was determined, which corresponds to effector memory T lymphocytes. With a moderately increased relative amount of this population, T lymphocytes 2 were usually represented by T helpers 2. A pronounced increase in the population was observed due to T cytotoxic lymphocytes 2. Regardless of the predominance of the Т helper or Т cytotoxic 2 cells in patients revealed an increase in spontaneous production of interleukin 4 at a normal level of interferon. An increase in the peripheral blood T lymphocytes with CD294 expression contributes to the development, maintenance and exacerbation of allergic inflammation with the participation of IgEdependent and IgE-independent mechanisms. The CD3+CD294+cell population should be determined as an additional parameter in assessing the presence of sensitization in the basophil activation test in patients with hypersensitivity reactions. The use of this laboratory biomarker to assess the dominant type of immune inflammation will make it possible to personalize the therapy of the examined patients. Identification of pronounced deviations of indicators from the average values of a population will influence the tactics of patient management.

Correlation of the prognostic value of FNDC4 in glioblastoma with macrophage polarization

BackgroundGlioblastoma is among the most malignant tumors in the central nervous system and characterized by strong invasion and poor prognosis. Fibronectin type III domain-containing 4 (FNDC4) plays various important roles in the human body, including participating in cellular metabolism and inflammatory responses to cardiovascular diseases, influencing immune cells, and exerting anti-inflammatory effects; however, the role of FNDC4 in glioblastoma has not been reported.MethodsIn this study, bioinformatics databases, including TCGA, CGGA, GTEx, and TIMER, were used to analyze the differential expression of FNDC4 genes and cell survival, in addition to investigating its relationship with immune cell infiltration. Additionally, we overexpressed FNDC4 in glioblastoma cell lines U87 and U251 by lentiviral transfection and detected changes in proliferation, cell cycle progression, and apoptosis. Following collection of monocytes from the peripheral blood of healthy individuals and transformation into M0 macrophages, we performed flow cytometry to detect the polarizing effect of exogenous FNDC4, as well as the effect of FNDC4-overexpressing glioblastoma cells on macrophage polarization in a co-culture system.ResultsWe identified that significantly higher FNDC4 expression in glioblastoma tissue relative to normal brain tissue was associated with worse prognosis. Moreover, we found that FNDC4 overexpression in U87 and U251 cells resulted in increased proliferation and affected the S phase of tumor cells, whereas cell apoptosis remained unchanged. Furthermore, exogenous FNDC4 inhibited the M1 polarization of M0 macrophages without affecting M2 polarization; this was also observed in glioblastoma cells overexpressing FNDC4.ConclusionsFNDC4 expression is elevated in glioblastoma, closely associated with poor prognosis, and promoted the proliferation of glioblastoma cells, affected the S phase of tumor cells while inhibiting macrophage polarization.

Presence of innate lymphoid cells in allogeneic hematopoietic grafts correlates with reduced graft-versus-host disease

BackgroundAllogeneic hematopoietic cell transplantation (HCT) can be devastating when graft-versus-host disease (GvHD) develops. GvHD is characterized by mucosal inflammation due to breaching of epithelial barriers. Innate lymphoid cells (ILCs) are immune modulatory cells that are important in the maintenance of epithelial barriers, via their production of interleukin (IL)-22 and their T cell suppressive properties. After chemo- and radiotherapy, ILCs are depleted, and recovery after remission-induction therapy and after allogeneic HCT is slow and incomplete in a significant number of patients, which is associated with an increased risk to develop acute GvHD. ObjectiveTo investigate whether the presence of mature ILCs within G-CSF–mobilized HCT grafts is correlated with the development of acute GvHD after allogeneic HCT. Study DesignWe analyzed ILCs in a cohort of 36 patients who received allogeneic HCT for a hematologic malignancy, by flow-cytometric immune-phenotyping of prospectively collected, cryopreserved peripheral blood mononuclear cells (PBMCs) and donor-derived HCT grafts collected for the same patients. Biased analysis, with ILCs defined as CD3−lineage−CD45+CD127+CD161+ lymphocytes, was performed using FlowJo version 10 software. Unbiased analysis was done using FlowSOM, which uses a self-organizing map (SOM) with a minimal spanning tree (MST) to define and visualize different clusters present in the samples. ResultsRemission-induction therapy significantly depleted ILCs from the blood, and patients who had a relatively low percentage of ILCs before allogeneic HCT were significantly more prone to develop acute GvHD, confirming previous findings in a separate cohort. Allogeneic HCT grafts, which were all obtained from the blood of G-CSF–mobilized healthy donors, contained ILCs at a frequency very similar to the peripheral blood of healthy individuals. The ILC subset composition was also comparable to that of the blood of healthy individuals, with the exception of NKp44+ ILC3s, which were significantly more abundant in HCT grafts. The relative ILC content of the graft tended to correlate with ILC reconstitution after allogeneic HCT, suggesting that peripheral expansion of transplanted mature ILCs may contribute to early ILC reconstitution after allogeneic HCT. Patients who received a relatively ILC-poor HCT graft had a significantly increased risk to develop acute GvHD, compared with patients who received relatively ILC-rich allogeneic HCT grafts. Unbiased phenotypic analysis with the FlowSOM algorithm confirmed that allogeneic HCT grafts of patients who developed acute GvHD contained a lower frequency of ILCs that clustered in NKp44+ ILC3 signature groups. ConclusionThe presence of ILCs in allogeneic HCT grafts is associated with a reduced risk to develop acute GvHD. These data suggest that enhancement of ILC reconstitution of ILC3s in particular, for example via adoptive transfer of ILCs, may prevent acute GvHD and has the potential to improve outcome of allogeneic HCT recipients.

Comprehensive phenotyping of human peripheral blood B lymphocytes in healthy conditions

The B cell compartment provides innate and adaptive immune defenses against pathogens. Different B cell subsets, reflecting the maturation stages of B cells, have noninterchangeable functions and roles in innate and adaptive immune responses. In this review, we provide an overview of the B cell subsets present in peripheral blood of healthy individuals. A specific gating strategy is also described to clearly and univocally identify B cell subsets based on the their phenotypic traits by flow cytometric analysis.

Single-Cell RNA-Seq of T Cells in B-ALL Patients Reveals an Exhausted Subset with Remarkable Heterogeneity.

Characterization of functional T cell clusters is key to developing strategies for immunotherapy and predicting clinical responses in leukemia. Here, single‐cell RNA sequencing is performed with T cells sorted from the peripheral blood of healthy individuals and patients with B cell‐acute lymphoblastic leukemia (B‐ALL). Unbiased bioinformatics analysis enabled the authors to identify 13 T cell clusters in the patients based on their molecular properties. All 11 major T cell subsets in healthy individuals are found in the patients with B‐ALL, with the counterparts in the patients universally showing more activated characteristics. Two exhausted T cell populations, characterized by up‐regulation of TIGIT, PDCD1, HLADRA, LAG3, and CTLA4 are specifically discovered in B‐ALL patients. Of note, these exhausted T cells possess remarkable heterogeneity, and ten sub‐clusters are further identified, which are characterized by different cell cycle phases, naïve states, and GNLY (coding granulysin) expression. Coupled with single‐cell T cell receptor repertoire profiling, diverse originations of the exhausted T cells in B‐ALL are suggested, and clonally expanded exhausted T cells are likely to originate from CD8+ effector memory/terminal effector cells. Together, these data provide for the first‐time valuable insights for understanding exhausted T cell populations in leukemia.

Calpain Participates in Cortical Cytoskeleton Modification and DNA Release during Neutrophil Extracellular Trap Formation

Introduction: The formation of neutrophil extracellular traps (NETs) is a process in which several kinds of enzymes participate generating posttranslational modifications of proteins. NETs have been associated with infectious, autoimmune, and inflammatory diseases. Inhibition of several proteases reduces the formation of NETs. In the present work, we analyzed the role of several broad-acting and specific inhibitors of proteases in the formation of NETs. Methods: Neutrophils were isolated from peripheral blood of healthy individuals by density gradient. The neutrophils were quantified and seeded into cell culture plates. Phorbol myristate acetate and A23187 were used as NETs inducers, and several specific inhibitors of proteases were used. The cells were stained for cytoskeleton or DNA. The cell-free supernatants were used to assess DNA release. Statistical analysis was carried out by a Kruskal-Wallis or ANOVA test. Results: We observed marked changes in actin organization after the induction of NETs, suggesting that the cytoskeleton is being actively regulated. When we used protease inhibitors, the release of DNA was reduced, suggesting the participation of actin remodeling in the process. Further characterization of the specific proteases revealed that calpain modulates the reorganization of actin cytoskeleton and DNA release. Preservation of part of the actin cytoskeleton suggests that DNA release is not only a mechanic process associated to the chromatin decondensation; rather the process is highly regulated by active proteases that promote cytoskeleton reorganization and chromatin decondensation that culminates in DNA release. Conclusion: Calpain mediates the DNA release in the NET formation process by the modification of cortical actin cytoskeleton in a calcium-dependent manner.

Machine Learning (ML) Can Successfully Support Microscopic Differential Counts of Peripheral Blood Smears in a High Throughput Hematology Laboratory

Background: Cytomorphology is the gold standard for quick assessment of peripheral blood and bone marrow samples in hematological neoplasms. It is a broadly-accepted method for orchestrating more specific diagnostics including immunophenotyping or genetics. Inter-/intra-observer-reproducibility of single cell classification is only 75 to 90%. Only a limited number of cells (100 - 500 cells/smear) is read in a time-consuming procedure. Machine learning (ML) is more reliable where human skills are limited, i.e. in handling large amounts of data or images. We here tested ML to differentiate peripheral blood leukocytes in a high throughput hematology laboratory. Aim: To establish an ML-based cell classifier capable of identifying healthy and pathologic cells in digitalized peripheral blood smear scans at an accuracy competitive with or outperforming human expert level. Methods: We selected >2,600 smears out of our unique archive of > 250,000 peripheral blood smears from hematological neoplasms. Depending on quality, we scanned up to 1,000 single cell images per smear. For image acquisition, a Metafer Scanning System (Zeiss Axio Imager.Z2 microscope, automatic slide feeder and automatic oiling device) from MetaSystems (Altlussheim, GER) was used. Areas of interest were defined by pre-scan in 10x magnification followed by high resolution scan in 40x to generate cell images for analysis. Average capture times for 300/500 cells were 3:43/4:37 min We set up a supervised ML-learning model using colour images (144x144 pixels) as input, outputting predicted probabilities of 21 predefined classes. We used ImageNet-pretrained Xception as our base model. We trained, evaluated and deployed the model using Amazon SageMaker on a subset of 82,974 images randomly selected from 514,183 cells captured and labelled for this study. 20 different cell types and one garbage class were classified. We included cell type categories referring to the critical importance of detecting rare leukemia subtypes (e.g. APL). Numbers of images from respective 21 classes ranged from 1,830 to 14,909 (median: 2,945). Minority classes were up-sampledto handle imbalances. Each picture was labelled by highly skilled technicians (median years practicing in this laboratory: 5) and two independent hematologists (median years at microscope: 20). Results: On a separate test set of 8,297 cells, our classifier was able to predict any of the five cell types occurring in the peripheral blood of healthy individuals (PMN, lymphocytes, monocytes, eosinophils, basophils) at very high median accuracy (97.0%) Median prediction accuracy of 15 rare or pathological cell types was 91.3%. For six critical pathological cell forms (myeloblasts, atypical/bilobulated promyelocytes in APL/APLv, hairy cells, lymphoma cells,plasma cells), median accuracy was 93.4% (sensitivity 93.8%). We saw a very high "T98 accuracy" for these cell types (98.5%) which is the accuracy of cell type predictions with prediction probability >0.98 (achieved in 2231/2417 cases), implicating that critical cells predicted with probability <0.98 should be flagged for human expert validation with priority. For all 21 classes median accuracy was 91.7%. Accuracy was lower for cells representing consecutive steps of maturation, e.g. promyelo-/myelo-/metamyelocytes, reproducing inconsistencies from the human-built phenotypic classification system (s.Fig.). Conclusions: We demonstrate an automated workflow using automatic microscopic cell capturing and ML-driven cell differentiation in samples of hematologic patients. Reproducibility, accuracy, sensitivity and specificity are above 90%, for many cell types above 98%. By flagging suspicious cells for humanvalidation, this tool can support even experienced hematology professionals, especially in detecting rare cell types. Given an appropriate scanning speed, it clearly outperforms human investigators in terms of examination time and number of differentiated cells. An ML-based intelligence can make its skills accessible to hematology laboratories on site or after upload of scanned cell images, independent of time/location. A cloud-based infrastructure is available. A prospective head to head challenge between ML-based classifier and human experts comparing sensitivity and accuracy for detection of all cell classes in peripheral blood will be tested to proof suitability for routine use (NCT 4466059). Figure Disclosures Heo: AWS: Current Employment. Wetton:AWS: Current Employment. Drescher:MetaSystems: Current Employment. Hänselmann:MetaSystems: Current Employment. Lörch:MetaSystems: Current equity holder in private company.

Transcriptomic Profiling of Human Effector and Regulatory T Cell Subsets Identifies Predictive Population Signatures.

After activation, CD4+ Th cells differentiate into functionally specialized populations that coordinate distinct immune responses and protect against different types of pathogens. In humans, these effector and memory Th cell subsets can be readily identified in peripheral blood based on their differential expression of chemokine receptors that govern their homeostatic and inflammatory trafficking. Foxp3+ regulatory T (Treg) cells can also be divided into subsets that phenotypically mirror each of these effector populations and share expression of key transcription factors and effector cytokines. In this study, we performed comprehensive transcriptional profiling of 11 phenotypically distinct Th and Treg cell subsets sorted from peripheral blood of healthy individuals. Despite their shared phenotypes, we found that mirror Th and Treg subsets were transcriptionally dissimilar and that Treg cell populations showed limited transcriptional diversity compared with Th cells. We identified core transcriptional signatures shared across all Th and Treg cell populations and unique signatures that define each of the Th or Treg populations. Finally, we applied these signatures to bulk Th and Treg RNA-sequencing data and found enrichment of specific Th and Treg cell populations in different human tissues. These results further define the molecular basis for the functional specialization and differentiation of Th and Treg cell populations and provide a new resource for examining Th and Treg specialization in RNA-sequencing data.

Eomes-Dependent Loss of the Co-activating Receptor CD226 Restrains CD8+ T Cell Anti-tumor Functions and Limits the Efficacy of Cancer Immunotherapy

CD8+ Tcells within the tumor microenvironment (TME) are exposed to various signals that ultimately determine functional outcomes. Here, we examined the role of the co-activating receptor CD226 (DNAM-1) in CD8+ Tcell function. The absence of CD226 expression identified a subset of dysfunctional CD8+ Tcells present in peripheral blood of healthy individuals. These cells exhibited reduced LFA-1 activation, altered TCR signaling, and a distinct transcriptomic program upon stimulation. CD226neg CD8+ Tcells accumulated in human and mouse tumors of diverse origin through an antigen-specific mechanism involving the transcriptional regulator Eomesodermin (Eomes). Despite similar expression of co-inhibitory receptors, CD8+ tumor-infiltratinglymphocyte failed to respond to anti-PD-1 in the absence of CD226. Immune checkpoint blockade efficacy was hampered in Cd226-/- mice. Anti-CD137 (4-1BB) agonists also stimulated Eomes-dependent CD226 loss that limited the anti-tumor efficacy of this treatment. Thus, CD226 loss restrains CD8+ Tcell function and limits the efficacy of cancer immunotherapy.

A minority of T cells recognizing tumor-associated antigens presented in self-HLA can provoke antitumor reactivity

AbstractTumor-associated antigens (TAAs) are monomorphic self-antigens that are proposed as targets for immunotherapeutic approaches to treat malignancies. We investigated whether T cells with sufficient avidity to recognize naturally overexpressed self-antigens in the context of self-HLA can be found in the T-cell repertoire of healthy donors. Minor histocompatibility antigen (MiHA)-specific T cells were used as a model, as the influence of thymic selection on the T-cell repertoire directed against MiHA can be studied in both self (MiHApos donors) and non-self (MiHAneg donors) backgrounds. T-cell clones directed against the HLA*02:01-restricted MiHA HA-1H were isolated from HA-1Hneg/HLA-A*02:01pos and HA-1Hpos/HLA-A*02:01pos donors. Of the 16 unique HA-1H–specific T-cell clones, five T-cell clones derived from HA-1Hneg/HLA-A*02:01pos donors and one T-cell clone derived from an HA-1Hpos/HLA-A*02:01pos donor showed reactivity against HA-1Hpos target cells. In addition, in total, 663 T-cell clones (containing at least 91 unique clones expressing different T-cell receptors) directed against HLA*02:01-restricted peptides of TAA WT1-RMF, RHAMM-ILS, proteinase-3-VLQ, PRAME-VLD, and NY-eso-1-SLL were isolated from HLA-A*02:01pos donors. Only 3 PRAME-VLD–specific and one NY-eso-1-SLL–specific T-cell clone provoked interferon-γ production and/or cytolysis upon stimulation with HLA-A*02:01pos malignant cell lines (but not primary malignant samples) naturally overexpressing the TAA. These results show that self-HLA–restricted T cells specific for self-antigens such as MiHA in MiHApos donors and TAAs are present in peripheral blood of healthy individuals. However, clinical efficacy would require highly effective in vivo priming by peptide vaccination in the presence of proper adjuvants or in vitro expansion of the low numbers of self-antigen–specific T cells of sufficient avidity to recognize endogenously processed antigen.

Different regulation of PARP1, PARP2, PARP3 and TRPM2 genes expression in acute myeloid leukemia cells

BackgroundAcute myeloid leukemia (AML) is a heterogenic lethal disorder characterized by the accumulation of abnormal myeloid progenitor cells in the bone marrow which results in hematopoietic failure. Despite various efforts in detection and treatment, many patients with AML die of this cancer. That is why it is important to develop novel therapeutic options, employing strategic target genes involved in apoptosis and tumor progression.MethodsThe aim of the study was to evaluate PARP1, PARP2, PARP3, and TRPM2 gene expression at mRNA level using qPCR method in the cells of hematopoietic system of the bone marrow in patients with acute myeloid leukemia, bone marrow collected from healthy patients, peripheral blood of healthy individuals, and hematopoietic stem cells from the peripheral blood after mobilization.ResultsThe results found that the bone marrow cells of the patients with acute myeloid leukemia (AML) show overexpression of PARP1 and PARP2 genes and decreased TRPM2 gene expression. In the hematopoietic stem cells derived from the normal marrow and peripheral blood after mobilization, the opposite situation was observed, i.e. TRPM2 gene showed increased expression while PARP1 and PARP2 gene expression was reduced. We observed positive correlations between PARP1, PARP2, PARP3, and TRPM2 genes expression in the group of mature mononuclear cells derived from the peripheral blood and in the group of bone marrow-derived cells. In AML cells significant correlations were not observed between the expression of the examined genes. In addition, we observed that the reduced expression of TRPM2 and overexpression of PARP1 are associated with a shorter overall survival of patients, indicating the prognostic significance of these genes expression in AML.ConclusionsOur research suggests that in physiological conditions in the cells of the hematopoietic system there is mutual positive regulation of PARP1, PARP2, PARP3, and TRPM2 genes expression. PARP1, PARP2, and TRPM2 genes at mRNA level deregulate in acute myeloid leukemia cells.

CD81+ Exosomes Play a Pivotal Role in the Establishment of Hepatitis C Persistent Infection and Contribute Toward the Progression of Hepatocellular Carcinoma.

CD81 serves as an immune modulator, playing its role in tumor growth and metastasis of hepatitis C virus (HCV)-mediated hepatocellular carcinoma (HCC). CD81 serves as a coreceptor of viral entry and is found to be enriched in exosomes. HCV E2 protein when associated with CD81 may be responsible for B cell lymphoproliferative disorders, as extrahepatic manifestation. Studies predict that HCV association with exosomes, leads to the establishment of persistent infection, through immune evasion. Herein, we confirm the association of HCV particles with CD81+ exosomes. Breifly, exosomes were enriched from peripheral blood of chronic HCV patients who have developed HCC. Sideways, exosomes were also enriched from peripheral blood of healthy individuals, who exhibited normal liver function test profile and had no known infection. Isolation of subpopulation of CD81+ exosomes was performed through immunocapture, followed by detection using FACS. Scanning electron microscopy confirmed the physical association of a fraction of exosome with HCV. CD81+ exosomes from chronic HCV patients with HCC were more granulated and larger when compared with those enriched from a healthy individual and HCV RNA was also detected in enriched fractions of CD81+ exosomes from HCV-positive HCC patients only, through real-time quantitative polymerase chain reaction. We concluded that CD81+ exosomes carry HCV particles and the association plays a pivotal role in establishing persistent infection, through immune evasion, thus leading to HCC progression. Exosomal CD81 and its interacting proteins might, therefore, serve as a potential prognostic marker and therapeutic target in HCV progression mediated by active HCV infection.