scholarly journals Whole blood‐based measurement of SARS‐CoV‐2‐specific T cells reveals asymptomatic infection and vaccine immunogenicity in healthy subjects and patients with solid organ cancers

Immunology ◽  
2021 ◽  
Author(s):  
Martin J. Scurr ◽  
Wioleta M. Zelek ◽  
George Lippiatt ◽  
Michelle Somerville ◽  
Stephanie E. A. Burnell ◽  
...  
Keyword(s):  
T Cells ◽  
Whole Blood ◽  
Healthy Subjects ◽  
Asymptomatic Infection ◽  
Solid Organ ◽  
Vaccine Immunogenicity

Activation and Effector Phenotype of CD8+ T Cells Crossprimed by a Minor Histocompatibility Antigen Expressed on Red Blood Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2571-2571
Author(s):  
Maxime Desmarets ◽  
James C. Zimring
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Whole Blood ◽  
Granzyme B ◽  
Solid Organ ◽  
Minor Histocompatibility Antigens ◽  
Viable Solution ◽  
Cfse Dilution ◽  
A Minor

Abstract Background: Transfusion immunology research has traditionally focused on humoral immunity to RBC antigens, with study of CD4+ T cell and B cell responses. However, minor histocompatibility antigens (mHAs) on transfused RBCs in theory can also be cross-presented by recipient APCs to CD8+ T cells. This has clinical relevance in the context of transfusion patients who undergo transplantation, as several lines of evidence suggest that immunization to mHAs can lead to rejection of bone marrow and solid organ transplants. To test the hypothesis that mHAs on RBCs are capable of inducing immune responses through cross priming of CD8+ T cells, we created a novel transgenic mouse (HOD mouse), which expresses Ovalbumin (OVA) on RBCs but neither on leukocytes nor platelets. Methods: 1×107 OT-I splenocytes (TCR transgenic for the OVA derived peptide SIINFEKL presented by H-2Kb) were adoptively transferred into B6.Thy1.1 mice. 100μl of packed HOD RBCs from whole or leukoreduced blood were then transfused intravenously. Control mice were transfused with FVB whole blood (HOD mice are on a FVB background). Three to 4 days post transfusion the OT-I T cells were visualized in the spleen by staining with anti-CD8 antibody and the Kb/SIINFEKL tetramer. Proliferation was evaluated by CFSE dilution. The activation phenotype was studied by staining with antibodies against CD44 and CD62L. Expression of effector molecules was studied by staining with antibodies against CD107a, Granzyme B, IFNγ, and IL-2. Results: Compared to background analysis of OT-I T cells in control mice that received FVB blood, OT-I T cells in HOD recipients had a maximal expansion 24 fold higher in mice transfused with HOD leukoreduced blood (p=.0001), and 26 fold in mice transfused with HOD whole blood (p=.0672). Compared to naïve OT-I T cells, cross-primed OT-I T cells were CD44high, CD62Llow, CD107ahigh, suggesting activation and degranulation. Expression of IFNγ was also increased. However, cross-primed OT-I T cells were Granzyme Blow and did not have increased IL-2 expression. Lack of increased Granzyme B and IL-2 was not an artifact of the assay not working, as strong expression of both Granzyme B and IL-2 were detected in OT-I T cells stimulated with OVA in vitro. Discussion: Overall these results demonstrate that RBC antigens can be cross-primed into the MHC class I pathway by recipient APCs. However, activation of CD8+ T cells specific for the RBC antigen demonstrates differentiation into an incomplete effector phenotype. The effect is unlikely due to contaminating donor leukocytes in the transfused unit since OVA is not expressed by WBCs. Moreover, we did not see any differences in proliferation or activation phenotype between the mice transfused with whole blood and leukoreduced blood. Activation was not due to direct presentation by residual cells, as the HOD donor mice are on an FVB (H-2q) background and cannot directly present mHAs to the H-2b restricted OT-I T cells. The reason for an incomplete effector profile is unclear, but may be because transfused RBCs are a sterile introduction of foreign antigen. These results also suggest better leukoreduction is not a viable solution to the problem of transfusion-induced immunization to mHAs, as the antigen is coming from the RBCs themselves. Matching for relevant mHAs or use of immunomodulatory treatments may be required.

scholarly journals Comparison of Standardized Cytomegalovirus (CMV) Viral Load Thresholds in Whole Blood and Plasma of Solid Organ and Hematopoietic Stem Cell Transplant Recipients with CMV Infection and Disease

2017 ◽  
Vol 4 (3) ◽  
Author(s):  
M Veronica Dioverti ◽  
Brian D Lahr ◽  
Jeffrey J Germer ◽  
Joseph D Yao ◽  
Michelle L Gartner ◽  
...  
Keyword(s):  
Stem Cell ◽  
Viral Load ◽  
Hematopoietic Stem Cell ◽  
Whole Blood ◽  
Asymptomatic Infection ◽  
Solid Organ ◽  
Cell Transplant ◽  
Transplant Recipients ◽  
Hematopoietic Stem ◽  
Cmv Disease

Abstract Background Quantification of cytomegalovirus (CMV) deoxyribonucleic acid (DNA) has important diagnostic, prognostic, and therapeutic implications in the management of transplant recipients. We aimed to assess a viral load in plasma and whole blood that distinguishes CMV disease from asymptomatic infection in a cohort of solid organ and hematopoietic stem cell transplantation. Methods We prospectively measured and compared CMV viral load in paired plasma and whole blood samples collected from transplant recipients with CMV infection and disease. Cytomegalovirus viral loads were determined by a commercially available US Food and Drug Administration-approved quantitative assay (COBAS AmpliPrep/COBAS TaqMan CMV Test [CAP/CTM CMV]) calibrated to the first World Health Organization International Standard for CMV DNA quantification. Results Moderate agreement of CMV viral load was observed between plasma and whole blood, with 31% of samples having discordant findings, particularly among samples with low DNA levels. Among the subset of samples where both paired samples had quantifiable levels, we observed a systematic bias that reflected higher viral load in whole blood compared with plasma. Based on receiver operating curve analysis, an initial plasma CMV viral load threshold of 1700 IU/mL in solid organ transplant recipients (sensitivity 80%, specificity 74%) and 1350 IU/mL in allogeneic hematopoietic stem cell transplant recipients (sensitivity 87%, specificity 87%) distinguished CMV disease and asymptomatic infection. Conclusions This study identifies standardized viral load thresholds that distinguish CMV disease from asymptomatic infection using CAP/CTM CMV assay. We propose these thresholds as potential triggers to be evaluated in prospective studies of preemptive therapy. Plasma was better than whole blood for measuring viral load using the CAP/CTM CMV assay.

Stability of antioxidant vitamins in whole human blood during overnight storage at 4°C and frozen storage up to 6 months

2018 ◽  
Vol 88 (3-4) ◽  
pp. 151-157 ◽  
Author(s):  
Scott W. Leonard ◽  
Gerd Bobe ◽  
Maret G. Traber
Keyword(s):  
Ascorbic Acid ◽  
Whole Blood ◽  
Healthy Subjects ◽  
Frozen Storage ◽  
Blood Collection ◽  
Plasma Samples ◽  
Optimal Conditions ◽  
Plasma Ascorbic Acid ◽  
Blood Samples ◽  
Sample Handling

Abstract. To determine optimal conditions for blood collection during clinical trials, where sample handling logistics might preclude prompt separation of erythrocytes from plasma, healthy subjects (n=8, 6 M/2F) were recruited and non-fasting blood samples were collected into tubes containing different anticoagulants (ethylenediaminetetra-acetic acid (EDTA), Li-heparin or Na-heparin). We hypothesized that heparin, but not EDTA, would effectively protect plasma tocopherols, ascorbic acid, and vitamin E catabolites (α- and γ-CEHC) from oxidative damage. To test this hypothesis, one set of tubes was processed immediately and plasma samples were stored at −80°C, while the other set was stored at 4°C and processed the following morning (~30 hours) and analyzed, or the samples were analyzed after 6 months of storage. Plasma ascorbic acid, as measured using HPLC with electrochemical detection (LC-ECD) decreased by 75% with overnight storage using EDTA as an anticoagulant, but was unchanged when heparin was used. Neither time prior to processing, nor anticoagulant, had any significant effects upon plasma α- or γ-tocopherols or α- or γ-CEHC concentrations. α- and γ-tocopherol concentrations remained unchanged after 6 months of storage at −80°C, when measured using either LC-ECD or LC/mass spectrometry. Thus, refrigeration of whole blood at 4°C overnight does not change plasma α- or γ-tocopherol concentrations or their catabolites. Ascorbic acid is unstable in whole blood when EDTA is used as an anticoagulant, but when whole blood is collected with heparin, it can be stored overnight and subsequently processed.

scholarly journals OP0021 IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN EARLY RHEUMATOID ARTHRITIS PATIENTS RESPONDING TO TOCILIZUMAB

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 12.2-12
Author(s):  
I. Muller ◽  
M. Verhoeven ◽  
H. Gosselt ◽  
M. Lin ◽  
T. De Jong ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Gene Expression ◽  
T Cells ◽  
Gene Ontology ◽  
Regulatory T Cells ◽  
Early Rheumatoid Arthritis ◽  
Whole Blood ◽  
Differentially Expressed ◽  
Rna Seq ◽  
Double Blind

Background:Tocilizumab (TCZ) is a monoclonal antibody that binds to the interleukin 6 receptor (IL-6R), inhibiting IL-6R signal transduction to downstream inflammatory mediators. TCZ has shown to be effective as monotherapy in early rheumatoid arthritis (RA) patients (1). However, approximately one third of patients inadequately respond to therapy and the biological mechanisms underlying lack of efficacy for TCZ remain elusive (1). Here we report gene expression differences, in both whole blood and peripheral blood mononuclear cells (PBMC) RNA samples between early RA patients, categorized by clinical TCZ response (reaching DAS28 < 3.2 at 6 months). These findings could lead to identification of predictive biomarkers for TCZ response and improve RA treatment strategies.Objectives:To identify potential baseline gene expression markers for TCZ response in early RA patients using an RNA-sequencing approach.Methods:Two cohorts of RA patients were included and blood was collected at baseline, before initiating TCZ treatment (8 mg/kg every 4 weeks, intravenously). DAS28-ESR scores were calculated at baseline and clinical response to TCZ was defined as DAS28 < 3.2 at 6 months of treatment. In the first cohort (n=21 patients, previously treated with DMARDs), RNA-sequencing (RNA-seq) was performed on baseline whole blood PAXgene RNA (Illumina TruSeq mRNA Stranded) and differential gene expression (DGE) profiles were measured between responders (n=14) and non-responders (n=7). For external replication, in a second cohort (n=95 therapy-naïve patients receiving TCZ monotherapy), RNA-seq was conducted on baseline PBMC RNA (SMARTer Stranded Total RNA-Seq Kit, Takara Bio) from the 2-year, multicenter, double-blind, placebo-controlled, randomized U-Act-Early trial (ClinicalTrials.gov identifier: NCT01034137) and DGE was analyzed between 84 responders and 11 non-responders.Results:Whole blood DGE analysis showed two significantly higher expressed genes in TCZ non-responders (False Discovery Rate, FDR < 0.05): urotensin 2 (UTS2) and caveolin-1 (CAV1). Subsequent analysis of U-Act-Early PBMC DGE showed nine differentially expressed genes (FDR < 0.05) of which expression in clinical TCZ non-responders was significantly higher for eight genes (MTCOP12, ZNF774, UTS2, SLC4A1, FECH, IFIT1B, AHSP, and SPTB) and significantly lower for one gene (TND2P28M). Both analyses were corrected for baseline DAS28-ESR, age and gender. Expression of UTS2, with a proposed function in regulatory T-cells (2), was significantly higher in TCZ non-responders in both cohorts. Furthermore, gene ontology enrichment analysis revealed no distinct gene ontology or IL-6 related pathway(s) that were significantly different between TCZ-responders and non-responders.Conclusion:Several genes are differentially expressed at baseline between responders and non-responders to TCZ therapy at 6 months. Most notably, UTS2 expression is significantly higher in TCZ non-responders in both whole blood as well as PBMC cohorts. UTS2 could be a promising target for further analyses as a potential predictive biomarker for TCZ response in RA patients in combination with clinical parameters (3).References:[1]Bijlsma JWJ, Welsing PMJ, Woodworth TG, et al. Early rheumatoid arthritis treated with tocilizumab, methotrexate, or their combination (U-Act-Early): a multicentre, randomised, double-blind, double-dummy, strategy trial. Lancet. 2016;388(10042):343-55.[2]Bhairavabhotla R, Kim YC, Glass DD, et al. Transcriptome profiling of human FoxP3+ regulatory T cells. Human Immunology. 2016;77(2):201-13.[3]Gosselt HR, Verhoeven MMA, Bulatovic-Calasan M, et al. Complex machine-learning algorithms and multivariable logistic regression on par in the prediction of insufficient clinical response to methotrexate in rheumatoid arthritis. Journal of Personalized Medicine. 2021;11(1).Disclosure of Interests:None declared

scholarly journals Severely ill COVID-19 patients display impaired exhaustion features in SARS-CoV-2-reactive CD8+ T cells

2021 ◽  
Vol 6 (55) ◽  
pp. eabe4782 ◽  
Author(s):  
Anthony Kusnadi ◽  
Ciro Ramírez-Suástegui ◽  
Vicente Fajardo ◽  
Serena J Chee ◽  
Benjamin J Meckiff ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Healthy Subjects ◽  
Single Cell Analysis ◽  
Severe Disease ◽  
T Cell Response ◽  
Severe Illness ◽  
Syncytial Virus ◽  
Apoptotic Pathways

The molecular properties of CD8+ T cells that respond to SARS-CoV-2 infection are not fully known. Here, we report on the single-cell transcriptomes of >80,000 virus-reactive CD8+ T cells, obtained using a modified Antigen-Reactive T cell Enrichment (ARTE) assay, from 39 COVID-19 patients and 10 healthy subjects. COVID-19 patients segregated into two groups based on whether the dominant CD8+ T cell response to SARS-CoV-2 was ‘exhausted’ or not. SARS-CoV-2-reactive cells in the exhausted subset were increased in frequency and displayed lesser cytotoxicity and inflammatory features in COVID-19 patients with mild compared to severe illness. In contrast, SARS-CoV-2-reactive cells in the dominant non-exhausted subset from patients with severe disease showed enrichment of transcripts linked to co-stimulation, pro-survival NF-κB signaling, and anti-apoptotic pathways, suggesting the generation of robust CD8+ T cell memory responses in patients with severe COVID-19 illness. CD8+ T cells reactive to influenza and respiratory syncytial virus from healthy subjects displayed polyfunctional features and enhanced glycolysis. Cells with such features were largely absent in SARS-CoV-2-reactive cells from both COVID-19 patients and healthy controls non-exposed to SARS-CoV-2. Overall, our single-cell analysis revealed substantial diversity in the nature of CD8+ T cells responding to SARS-CoV-2.

Low-dose of tacrolimus favors the induction of functional CD4+CD25+FoxP3+ regulatory T cells in solid-organ transplantation

2009 ◽  
Vol 9 (5) ◽  
pp. 564-569 ◽  
Author(s):  
Zhen Wang ◽  
Bingyi Shi ◽  
Hailong Jin ◽  
Li Xiao ◽  
Yongwei Chen ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Organ Transplantation ◽  
Low Dose ◽  
Solid Organ Transplantation ◽  
Solid Organ

scholarly journals Unique Gene Expression Patterns in Human T-cell Lines Generated from Multiple Sclerosis Patients by Stimulation with a Synthetic MOG Peptide

2005 ◽  
Vol 12 (3) ◽  
pp. 203-209 ◽  
Author(s):  
Mathilda Mandel ◽  
Michael Gurevich ◽  
Gad Lavie ◽  
Irun R. Cohen ◽  
Anat Achiron
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Healthy Subjects ◽  
Expression Patterns ◽  
Gene Expression Pattern ◽  
Gene Expression Patterns ◽  
T Cell Lines ◽  
Myelin Antigens

Multiple sclerosis (MS) is an autoimmune disease where T-cells activated against myelin antigens are involved in myelin destruction. Yet, healthy subjects also harbor T-cells responsive to myelin antigens, suggesting that MS patient-derived autoimmune T-cells might bear functional differences from T-cells derived from healthy individuals. We addressed this issue by analyzing gene expression patterns of myelin oligodendrocytic glycoprotein (MOG) responsive T-cell lines generated from MS patients and healthy subjects. We identified 150 transcripts that were differentially expressed between MS patients and healthy controls. The most informative 43 genes exhibited >1.5-fold change in expression level. Eighteen genes were up-regulated including BCL2, lifeguard, IGFBP3 and VEGF. Twenty five genes were down-regulated, including apoptotic activators like TNF and heat shock protein genes. This gene expression pattern was unique to MOG specific T-cell lines and was not expressed in T-cell lines reactive to tetanus toxin (TTX). Our results indicate that activation in MS that promotes T-cell survival and expansion, has its own state and that the unique gene expression pattern that characterize autoreactive T-cells in MS represent a constellation of factors in which the chronicity, timing and accumulation of damage make the difference between health and disease.

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