scholarly journals Reactive T Cells in Convalescent COVID-19 Patients With Negative SARS-CoV-2 Antibody Serology

2021 ◽  
Vol 12 ◽  
Author(s):  
Sophie Steiner ◽  
Tatjana Schwarz ◽  
Victor M. Corman ◽  
Franziska Sotzny ◽  
Sandra Bauer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell Response ◽  
Healthy Controls ◽  
Disease Course ◽  
Rt Pcr ◽  
Viral Control ◽  
Specific Antibodies ◽  
Mild Disease

Despite RT-PCR confirmed COVID-19, specific antibodies to SARS-CoV-2 spike are undetectable in serum in approximately 10% of convalescent patients after mild disease course. This raises the question of induction and persistence of SARS-CoV-2-reactive T cells in these convalescent individuals. Using flow cytometry, we assessed specific SARS-CoV-2 and human endemic coronaviruses (HCoV-229E, -OC43) reactive T cells after stimulation with spike and nucleocapsid peptide pools and analyzed cytokine polyfunctionality (IFNγ, TNFα, and IL-2) in seropositive and seronegative convalescent COVID-19 patients as well as in unexposed healthy controls. Stimulation with SARS-CoV-2 spike and nucleocapsid (NCAP) as well as HCoV spike peptide pools elicited a similar T cell response in seropositive and seronegative post COVID-19 patients. Significantly higher frequencies of polyfunctional cytokine nucleocapsid reactive CD4+ T cells (triple positive for IFNγ, TNFα, and IL-2) were observed in both, seropositive (p = 0.008) and seronegative (p = 0.04), COVID-19 convalescent compared to healthy controls and were detectable up to day 162 post RT-PCR positivity in seronegative convalescents. Our data indicate an important role of NCAP-specific T cells for viral control.

The Inhibitory Role of Lactacystin and β-Lactacystin on T-cell Activation and Proliferation

2004 ◽  
Vol 36 (2) ◽  
pp. 123-127 ◽  
Author(s):  
Peng-Hong Song ◽  
Hai-Yang Xie ◽  
Shu-Sen Zheng ◽  
Jian Wu
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
T Cell Activation ◽  
Cell Activation ◽  
Proteasome Inhibitors ◽  
Rt Pcr ◽  
Down Regulation

Abstract To evaluate the effects of proteasome inhibitors lactacystin (LAC) and β-lactacystin (β-LAC) on the proliferation and activation of T lymphocytes, flow cytometry was used to analyze the proliferation and the expression of CD69, CD25 and CD3 of T lymphocytes activated by PHA. Furthermore, the expressions of PA28 and IL-2 mRNA were assayed by competitive RT-PCR. The results indicated that: (1) LAC and β-LAC significantly decreased the incorporation of BrdU and inhibited T lymphocytes proliferation in T lymphocytes activated by PHA; (2) although LAC and β-LAC did not affect the expression of CD69 at any time, they significantly inhibited the expression of CD25 (48 h, 72 h, P<0.05); (3) in comparison with control, LAC and β-LAC significantly down-regulated the expression of PA28 and IL-2 mRNA (48 h, 72 h, P<0.05). LAC and β-LAC significantly inhibited the proliferation and activation of T cells. Mechanisms involved are inhibition of CD25 and down-regulation of PA28 and IL-2 mRNA expressions.

scholarly journals Absence of Bim sensitizes mice to experimental Trypanosoma cruzi infection

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Marcela Hernández-Torres ◽  
Rogério Silva do Nascimento ◽  
Monica Cardozo Rebouças ◽  
Alexandra Cassado ◽  
Kely Catarine Matteucci ◽  
...  
Keyword(s):  
T Cells ◽  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Parasite Load ◽  
Peritoneal Macrophages ◽  
T Cell Response ◽  
No Production ◽  
Similar Reduction ◽  
Ifn Γ

AbstractChagas disease is a life-threatening disorder caused by the protozoan parasite Trypanosoma cruzi. Parasite-specific antibodies, CD8+ T cells, as well as IFN-γ and nitric oxide (NO) are key elements of the adaptive and innate immunity against the extracellular and intracellular forms of the parasite. Bim is a potent pro-apoptotic member of the Bcl-2 family implicated in different aspects of the immune regulation, such as negative selection of self-reactive thymocytes and elimination of antigen-specific T cells at the end of an immune response. Interestingly, the role of Bim during infections remains largely unidentified. To explore the role of Bim in Chagas disease, we infected WT, Bim+/−, Bim−/− mice with trypomastigotes forms of the Y strain of T. cruzi. Strikingly, our data revealed that Bim−/− mice exhibit a delay in the development of parasitemia followed by a deficiency in the control of parasite load in the bloodstream and a decreased survival compared to WT and Bim+/− mice. At the peak of parasitemia, peritoneal macrophages of Bim−/− mice exhibit decreased NO production, which correlated with a decrease in the pro-inflammatory Small Peritoneal Macrophage (SPM) subset. A similar reduction in NO secretion, as well as in the pro-inflammatory cytokines IFN-γ and IL-6, was also observed in Bim−/− splenocytes. Moreover, an impaired anti-T. cruzi CD8+ T-cell response was found in Bim−/− mice at this time point. Taken together, our results suggest that these alterations may contribute to the establishment of a delayed yet enlarged parasitic load observed at day 9 after infection of Bim−/− mice and place Bim as an important protein in the control of T. cruzi infections.

scholarly journals POS0776 CORRELATION OF PERIPHERAL CD4+GRANZB+CTLS WITH DISEASE SEVERITY IN PATIENTS WITH PRIMARY SJÖGREN’S SYNDROME

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 641.3-642
Author(s):  
Q. Wang ◽  
L. Gu ◽  
M. Zhang
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Sjögren’S Syndrome ◽  
Mhc Class Ii ◽  
Sjögren's Syndrome ◽  
Sjogren’S Syndrome ◽  
Sjogren's Syndrome ◽  
Healthy Controls ◽  
Extraglandular Manifestations ◽  
Sjögren‘S Syndrome

Background:Sjögren’s syndrome (SS) is a chronic autoimmune disorder. The major histopathologic lesion of it is a focal lymphocytic infiltrate around ductal and acinar epithelial cells, which include a majority of CD4+T. Several studies have shown that the epithelial cells in SS present diverse phenomena, such as MHC class II overexpression. CD4+T cells with cytotoxic activity (CD4 CTL) have been detected in various immune responses. They are characterized by their ability to secrete perforin and granzyme B to kill the target cells in an MHC class II-restricted fashion.Objectives:So this study was to investigate the correlation of peripheral CD4+GranzB+CTLs with disease severity and organ involvement in patients with primary Sjögren’s syndrome.Methods:We recruited 116 pSS patients and 46 healthy controls using flow cytometry to examine proportion of CD4+GranzB+CTLs in their peripheral blood, and immunofluorescence to test the expression of CD4+GranzB+CTLs in labial gland. The correlations of CD4+GranzB+CTLs and the relevant clinical data were analyzed.Results:We analyzed the percentage of CD4+GranzB+cytotoxic T cells in peripheral blood mononuclear cells (PBMCs) by flow cytometry. Frequency of peripheral CD4+GranzB+CTLs were measured in 116 patients with pSS and 46 healthy controls matched for age and sex. The percentage of CD4+GranzB+CTLs were significantly up-regulated in pSS patients than healthy controls (7.1%±4.9% vs 3.1%±1.9%, p <0.0001) and positive correlation with ESSDAI in pSS patients(r = 0.6332, p<0.001). The percentage of CD4+GranzB+CTLs were markedly higher in pSS patients with extraglandular manifestations. Moreover, CD4+GranzB+CTLs were observed in the lymphocytic foci and periductal areas of the LSGs and were elevated with increased foci index (FI). After excluding the other risk factors associated with pSS, CD4+GranzB+CTLs were still related to ESSDIA and extraglandular manifestations independently(p<0.05). ROC curve analysis indicated that the area under the curve (AUC) of CD4+GranzB+CTLs was 0.796 to predict the activity of pSS, and 0.851 to presume extraglandular manifestations. The best diagnostic cut-off point was 4.865 for pSS patients.Conclusion:In this study, We provide new evidence indicating involvement of CD4+GranzB+CTLs over activation in the disease pathophysiology of pSS, which may serve as a new biomarker to evaluate the activity and severity of pSS.References:[1]Takeuchi A, Saito T. Front Immunol. (2017) 23:194.[2]Brown DM, et al. Front Immunol. (2016) 9:93.[3]Polihronis M, et al. Clin Exp Immunol. (1998) 114:485-90.[4]Xanthou G, et al. Clin Exp Immunol. (1999) 118:154-63.[5]Maehara T, et al. Ann Rheum Dis. (2017) 76:377-385.[6]Goules AV, et al. Clin Immunol. (2017) 182:30-40.[7]Hashimoto K, et al. Proc Natl Acad Sci U S A. (2019) 116:24242-24251.[8]Croia C, et al. Arthritis Rheumatol. (2014) 66:2545-57.[9]Schmidt D,et al. J Clin Invest. (1996) 97:2027–37.[10]Pandya JM, et al. Arthritis Rheum. (2010) 62:3457–66.[11]Moosig F, et al. Clin Exp Immunol. (1998) 114:113–8.[12]Peeters LM, et al. Front Immunol. (2017) 20:1160.Table 1.Multivariate analysis of CD4+GranzB+CTLs influenced by pSS-related factorsregression coefficientstandard errort-statisticsp value95%CICD8+GranzB+CTLs(%)0.1440.0334.3346.9E-50.077, 0.211ESSDAI0.2560.1222.0950.0410.011, 0.502extraglandular manifestations2.6121.2682.0590.0450.065, 5.158Figure 1.Receiver operating characteristic (ROC) curve of the frequency of CD4+GranzB+CTLs to predict ESSDAI and extraglandular manifestations responseDisclosure of Interests:None declared

Role of erythrocytes and platelets in the hypercoagulable status in polycythemia vera through phosphatidylserine exposure and microparticle generation

2013 ◽  
Vol 109 (06) ◽  
pp. 1025-1032 ◽  
Author(s):  
Chunyan Gao ◽  
Xue Yang ◽  
Jianan Li ◽  
Wei Wang ◽  
Jinxiao Hou ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Blood Cells ◽  
Significant Inhibition ◽  
Polycythaemia Vera ◽  
Factor V ◽  
Healthy Controls ◽  
Clotting Time ◽  
Thrombotic Risk ◽  
Few Data

SummaryThe development of thrombosis in polycythaemia vera (PV) involves multifactorial processes including pathological activation of blood cells. Release of microparticles (MPs) by activated cells in diseases is associated with thrombotic risk, but relatively few data are available in PV. The aim of the present study was to investigate the increase in MP release and exposure of phosphatidylserine (PS) on the outer membrane of MP-origin cells in patients with PV, and to analyse their procoagulant activity (PCA). PS-positive MPs and cells were detected by flow cytometry, while PCA was assessed with clotting time and purified coagulation complex assays. We found that PV patients had elevated circulating lactadherin+ MPs, which mostly originating from erythrocytes, platelets, granulocytes, and endothelial cells, as well as increased PS exposing erythrocytes/platelets as compared to secondary polycythaemia patients or healthy controls. These PS-bearing MPs and cells were highly procoagulant. Moreover, lactadherin competed factor V and VIII to PS and inhibited about 90% of the detected PCA in a dose-response manner while anti-TF antibody did no significant inhibition. Treatment with hydroxyurea is associated with a decrease in PS exposure and lactadherin+ MP release of erythrocytes/platelets. Our data demonstrate that PV patients are characterised by increased circulating procoagulant MPs and PS exposing erythrocytes/platelets, which could contribute to the hypercoagulable state in these patients.

scholarly journals PD-1 expression on NK cells can be related to cytokine stimulation and tissue residency

2021 ◽  
Author(s):  
Arnika K Wagner ◽  
Nadir Kadri ◽  
Chris Tibbitt ◽  
Koen van de Ven ◽  
Sunitha Bagawath-Singh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Nk Cells ◽  
Mhc Class I ◽  
Sequencing Analysis ◽  
Single Cell Rna Sequencing ◽  
Cytokine Stimulation ◽  
Deficient Mice

ABSTRACTAlthough PD-1 was shown to be a hallmark of T cells exhaustion, controversial studies have been reported on the role of PD-1 on NK cells. Here, we found by flow cytometry and single cell RNA sequencing analysis that PD-1 can be expressed on MHC class I-deficient tumor-infiltrating NK cells in vivo. We also demonstrate distinct alterations in the phenotype of PD-1-deficient NK cells which in part could be attributed to a decrease in tumor-infiltrating NK cells in PD-1-deficient mice. NK cells from PD-1-deficient mice exhibited a more mature phenotype which might reduce their capacity to migrate and kill in vivo. Finally, our results demonstrate that PD-L1 molecules in membranes of PD-1-deficient NK cells migrate faster than in NK cells from wildtype mice, suggesting that PD-1 and PD-L1 form cis interactions with each other on NK cells.

scholarly journals Baseline T cell immune phenotypes predict virologic and disease control upon SARS-CoV infection

2020 ◽  
Author(s):  
Jessica B. Graham ◽  
Jessica L. Swarts ◽  
Sarah R. Leist ◽  
Alexandra Schäfer ◽  
Vineet D. Menachery ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Clinical Outcomes ◽  
Severe Disease ◽  
Collaborative Cross ◽  
Viral Control ◽  
Major Barrier ◽  
Mild Disease ◽  
Immune Correlates ◽  
Wide Range

AbstractThe COVID-19 pandemic has revealed that infection with SARS-CoV-2 can result in a wide range of clinical outcomes in humans, from asymptomatic or mild disease to severe disease that can require mechanical ventilation. An incomplete understanding of immune correlates of protection represents a major barrier to the design of vaccines and therapeutic approaches to prevent infection or limit disease. This deficit is largely due to the lack of prospectively collected, pre-infection samples from indiviuals that go on to become infected with SARS-CoV-2. Here, we utilized data from a screen of genetically diverse mice from the Collaborative Cross (CC) infected with SARS-CoV to determine whether circulating baseline T cell signatures are associated with a lack of viral control and severe disease upon infection. SARS-CoV infection of CC mice results in a variety of viral load trajectories and disease outcomes. Further, early control of virus in the lung correlates with an increased abundance of activated CD4 and CD8 T cells and regulatory T cells prior to infections across strains. A basal propensity of T cells to express IFNg and IL17 over TNFa also correlated with early viral control. Overall, a dysregulated, pro-inflammatory signature of circulating T cells at baseline was associated with severe disease upon infection. While future studies of human samples prior to infection with SARS-CoV-2 are required, our studies in mice with SARS-CoV serve as proof of concept that circulating T cell signatures at baseline can predict clinical and virologic outcomes upon SARS-CoV infection. Identification of basal immune predictors in humans could allow for identification of individuals at highest risk of severe clinical and virologic outcomes upon infection, who may thus most benefit from available clinical interventions to restrict infection and disease.SummaryWe used a screen of genetically diverse mice from the Collaborative Cross infected with mouse-adapted SARS-CoV in combination with comprehensive pre-infection immunophenotyping to identify baseline circulating immune correlates of severe virologic and clinical outcomes upon SARS-CoV infection.

Development and characterization of a HCMV multiantigen therapeutic vaccine for GBM using the UNITE platform.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e14565-e14565
Author(s):  
Amit Adhikari ◽  
Juliete Macauley ◽  
Yoshimi Johnson ◽  
Mike Connolly ◽  
Tim Coleman ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Mouse Model ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cd8 T Cell ◽  
T Cell Response

e14565 Background: Glioblastoma (GBM) is an aggressive form of brain cancer with a median survival of 15 months which has remained unchanged despite technological advances in the standard of care. GBM cells specifically express human cytomegalovirus (HCMV) proteins providing a unique opportunity for targeted therapy. Methods: We utilized our UNITE (UNiversal Intracellular Targeted Expression) platform to develop a multi-antigen DNA vaccine (ITI-1001) that codes for the HCMV proteins- pp65, gB and IE-1. The UNITE platform involves lysosomal targeting technology, fusing lysosome-associated protein 1 (LAMP1) with target antigens resulting in increased antigen presentation by MHC-I and II. ELISpot, flow cytometry and ELISA techniques were used to evaluate the vaccine immunogenicity and a syngeneic, orthotopic GBM mouse model that expresses HCMV proteins was used for efficacy studies. The tumor microenvironment studies were done using flow cytometry and MSD assay. Results: ITI-1001 vaccination showed a robust antigen-specific CD4 and CD8 T cell response in addition to a strong humoral response. Using GBM mouse model, therapeutic treatment of ITI-1001 vaccine resulted in ̃56% survival with subsequent long-term immunity. Investigating the tumor microenvironment showed significant CD4 T cell infiltration as well as enhanced Th1 and CD8 T cell activation. Regulatory T cells were also upregulated upon ITI-1001 vaccination and would be an attractive target to further improve this therapy. In addition, tumor burden negatively correlated with number of activated CD4 T cells (CD4 IFNγ+) reiterating the importance of CD4 activation in ITI-1001 efficacy and potentially identifying treatment responders and non-responders. Further characterization of these two groups showed high infiltration of CD3+, CD4+ and CD8+ T cells in responders compared with non- responders along with higher CD8 T cell activation. Conclusions: Thus, we show that vaccination with HCMV antigens using the ITI-1001-UNITE platform generates strong cellular and humoral immune responses, triggering significant anti-tumor activity that leads to enhanced survival in mice with GBM.

scholarly journals The protective role of miR-223 in sepsis-induced mortality

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Dan Liu ◽  
Zhiding Wang ◽  
Huijuan Wang ◽  
Feifei Ren ◽  
Yanqin Li ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cells ◽  
Protective Factor ◽  
Linear Regression Analysis ◽  
Multiple Linear Regression Analysis ◽  
Negative Control ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Jurkat T Cells

Abstract Lymphocyte apoptosis appears to play an important role in immunodysfunction in sepsis. We investigated the role of miR-223 in cell proliferation and apoptosis to identify potential target downstream proteins in sepsis. We recruited 143 patients with sepsis and 44 healthy controls from the Chinese PLA General Hospital. Flow cytometry was used to sort monocytes, lymphocytes, and neutrophils from fresh peripheral blood. A miR-223 mimic and inhibitor were used for transient transfection of Jurkat T cells. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to assess expression of the miRNAs in cells. Western blot analysis was performed to measure protein expression. We evaluated the cell cycle and apoptosis by using flow cytometry (FCM) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Expression of miR-223 was significantly higher in the survivor group than in the nonsurvivor group. Multiple linear regression analysis revealed that SOFA scores correlated negatively with miR-223 and monocyte counts, with β coefficients (95% CI) of − 0.048 (− 0.077, − 0.019) and − 47.707 (− 83.871, − 11.543), respectively. miR-223 expression also correlated negatively with the percentage of apoptosis in lymphocytes. The rate of apoptosis in the miR-223 mimic group was significantly lower than that of the negative control, with an adverse outcome observed in the miR-223 inhibitor group. We also found that miR-223 enhanced the proliferation of Jurkat T cells and that inhibiting miR-223 had an inhibitory effect on the G1/S transition. We conclude that miR-223 can serve as a protective factor in sepsis by reducing apoptosis and enhancing cell proliferation in lymphocytes by interacting with FOXO1. Potential downstream molecules are HSP60, HSP70, and HTRA.

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