scholarly journals Usefulness of the New Hematological Parameter: Reactive Lymphocytes RE-LYMP with Flow Cytometry Markers of Inflammation in COVID-19

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 82
Author(s):  
Elżbieta Rutkowska ◽  
Iwona Kwiecień ◽  
Katarzyna Kulik ◽  
Beata Chełstowska ◽  
Krzysztof Kłos ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Lymphocytes ◽  
Hematological Parameter ◽  
Virus Infections ◽  
Activation Markers ◽  
Virus Group ◽  
Markers Of Inflammation ◽  
Activated T Lymphocytes ◽  
The Times ◽  
Reactive Lymphocytes

Identification of patients with activation of the immune system which indicates the presence of infection is essential, especially in the times of the global coronavirus 2019 (COVID-19) pandemic. The aim of the present study was to evaluate the reactive lymphocytes (RE-LYMP) parameter in COVID-19 and to correlate it with activation lymphocytes markers by flow cytometry. The study group consisted of 40 patients: with COVID-19 infection (n = 20) and with others virus infections without COVID-19 (COVID-19(−) virus (n = 20)) and 20 healthy donors (HC). Blood count and flow cytometry were performed. The COVID-19(+) group had significantly lower RE-LYMP parameter than the COVID-19(−) virus group (5.45 vs. 11.05, p < 0.05). We observed higher proportion of plasmablasts in the COVID-19(+) and COVID-19(−) virus groups than HC (8.8 vs. 11.1 vs. 2.7, p < 0.05). In the COVID-19(+) there was a lower proportion of CD4+ CD38+ cells than in the other groups (significant differences between COVID-19(+) and COVID-19(−) virus groups). RE-LYMP correlated with activated T lymphocytes CD38+ and HLA-DR+ in the COVID-19(−) virus group, however in the COVID-19(+) group correlations with T lymphocytes CD25+ and CD45RO+ were observed. In summary the analysis of the RE-LYMP together with flow cytometric activation markers can be helpful in identifying and distinguishing patients with COVID-19(+) from other viruses and HC.

scholarly journals Immunosuppression in Human Peripheral Blood T Lymphocytes by Fluvastatin

2004 ◽  
Vol 36 (10) ◽  
pp. 649-655 ◽  
Author(s):  
Li-Hua Wu ◽  
Yun-Le Wan ◽  
Hai-Yang Xie ◽  
Wen-Jin Zhang ◽  
Shu-Sen Zheng
Keyword(s):  
T Lymphocytes ◽  
Human Peripheral Blood ◽  
Flow Cytometric Analysis ◽  
Immunosuppressive Effect ◽  
Activation Markers ◽  
Killer Cells ◽  
Cd69 Expression ◽  
Nfat Activation ◽  
Activated T Lymphocytes

Abstract To investigate the immunosuppressive effect of fluvastatin on the PHA-activated T lymphocytes. T lymphocytes were isolated from the blood of healthy volunteers, cell proliferation and the activation markers expression were examined by flow cytometric analysis. Cytokine secretion was assayed by ELISA. LDH-release assay was used to detect activity of killer cells. NFAT activation was evaluated by TransAMTM ELISA kit. Results were as following. (1) Whereas no modification in CD25 expression was seen, fluvastatin at 5 μM caused a lower level of CD69 expression, accompanied by an essential suppression on proliferation, IL-2 production and cytotoxicity development in PHA-stimulated T cells. However, the level of secreted IL-10 had no change, and the level of IL-4 even experienced a significant increase. (2) Combined with cyclosporine A (CsA), fluvastatin would further repress CD69 expression, cells proliferation and activity of killer cells, meanwhile significantly induced the secretion of IL-4 and IL-10. (3) Fluvastatin treatment also resulted in a strong inhibition of NFAT activation. In conclusion, partly involving the blockage of activation of NFAT, fluvastatin exhibited an immunosuppressive effect in vitro.

scholarly journals Pre-Clinical Trial to Evaluate the Efficacy of Delayed Administration of Ixazomib in the Prophylaxis of Chronic Graft-Versus-Host Disease

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4521-4521 ◽  
Author(s):  
Teresa Ramos ◽  
Alfonso Rodríguez-Gil ◽  
Melanie Nufer ◽  
María Victoria Barbado ◽  
Estefania Garcia Guerrero ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Lymphocytes ◽  
Graft Versus Host Disease ◽  
Activation Markers ◽  
Graft Versus Host ◽  
Post Transplant ◽  
Activated T Lymphocytes

Abstract Introduction: Although survival rates have improved over the years, chronic graft-versus-host disease (cGvHD) remains the most frequent and severe complication in the long term after allogeneic hematopoietic stem cell transplantation (allo-HSCT). All the strategies developed to reduce its incidence are based on procedures aimed to decrease the risk of acute GvHD that, consequently, can also reduce the risk of cGvHD, mainly using immunosuppression in the early post-transplant period. These strategies induce apoptosis of donor T lymphocytes, responsible for a/cGvHD but also for graft-versus-tumor response. In the present project, we have evaluated a new strategy aimed to reduce the risk of cGvHD by manipulating the immune response not in the early post-transplant period, but in later phases. cGvHD develops via a complex cellular and molecular network involving thymus damage and unusual antigen presentation leading to aberrant T- and B-cell reactions characterized by Th17/Tc17 differentiation, macrophage sequestration in tissue, alloantibody formation, and fibrosis. Most of these cell populations are dependent on NF-kB for their activation. Ixazomib is a second-generation proteasome inhibitor for oral administration, representing an ideal candidate for prophylaxis in GvHD. Objective: We propose to develop a murine model of cGVHD and progressive onset cGvHD to test the efficacy of delayed administration of ixazomib as a novel strategy to decrease the risk of cGvHD. Methods: For in vitro studies, peripheral blood mononuclear cells from healthy donors were stimulated in the presence of different concentrations of ixazomib. After 48h and 120h, apoptosis was analyzed, and activation markers were evaluated. For in vivo studies, a murine model of progressive cGvHD (pcGvHD - allowing the recipient to survive to mild aGvHD, thus favoring autoreactive T cells to expand and cause cGvHD) and a scleroderma model of cGvHD were used. Ixazomib at 0.75mg/Kg/twice weekly, 2X from day +21 in the pcGvHD, with or without cyclosporine A (CyA) at 5mg/Kg/day from day 0 until the end of the study. For scleroderma cGvHD group, 3mg/Kg/2X from day +30 was used up to 120 days post-transplant. Flow cytometry was used to evaluate the different lymphocyte populations in the different target organs of the GvHD. Results: In vitro results showed that activated T lymphocytes are sensitive to the proapoptotic effect of ixazomib (>100nM), while high concentrations of the drug are necessary to cause apoptosis in the non-activated cells (5000nM). Through CD27 and CD45 expression, we verified that ixazomib has a proapoptotic effect mainly on naïve and effector cells. We also verified a decrease in the activation markers of CD3+CD25+INF-γ+ cells (p˂0.01, 1000nM, n = 4). In the animal trial, the survival of the pcGvHD model mice treated with ixazomib was significantly higher as compared to untreated mice, with a significant decrease in the signs of pcGvHD (Figure 1A). In addition, the combination of CyA and ixazomib improved survival as compared to those mice receiving CyA or ixazomib alone as well as untreated controls (Figure 1B). In the scleroderma cGvHD model, we observed that the animals treated with ixazomib showed significantly less signs of GvHD (p˂0.0001) (Figure 2). Multiparametric flow cytometer analyses showed that in the pcGvHD model, mice treated with ixazomib had a decrease of effector CD4 T-cells in bone marrow (p=0.06) and spleen (p=0.015) when compared to untreated mice. Also an increase of CD19+ cells in the colon (p=0.04), liver (p=0.035), lymph nodes (LN) (p=0.037) and lungs (p=0.03) was observed. Regarding the regulatory T cells (Foxp3+), the treated mice had a significant increase in LN (p=0.02), Peyer patches (p=0.015) and thymus (p=0.028) as compared to untreated mice. Conclusion: In vitro studies show that ixazomib induces apoptosis on activated T lymphocytes and decreases the expression of activation markers. Our in vivo model indicates that the combination of CyA and ixazomib for the prevention of pcGvHD and cGVHD after allogeneic HSCT is promising and merits further investigation in clinical trials. Figure 1. Survival of pcGvHD. Figure 2. Score graphic of scleroderma cGvHD. BM - Bone marrow, mice that were transplanted with BM from BALBc mice (Syngeneic). Disclosures Ramos: Takeda Oncology: Research Funding.

HLA-G Transference From Multipotent Mesenchymal Stromal Cells to Activated T-Lymphocytes.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3674-3674
Author(s):  
Rodrigo A Panepucci ◽  
Felipe Saldanha-Araujo ◽  
Kelen C R Malmegrim ◽  
Fabio M Oliveira ◽  
Patricia V B Palma ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Lymphocytes ◽  
Confocal Microscopy ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Nuclear Staining ◽  
Activated T Cells ◽  
Activated T Lymphocytes ◽  
Membrane Bound

Abstract Abstract 3674 Poster Board III-610 HLA-G is a nonclassic human leukocyte antigen which is characterized by its limited variability, its highly tissue-specific expression and for its very distinct immunological role. Instead of triggering immune responses, HLA-G is exclusively inhibitory, suppressing immune cell functions. For instance, HLA-G is expressed by cells in sites considered immunologically privileged, such as the fetal cytotrophoblast, at the fetal–maternal interface, were it acts protecting the fetal tissue from the mother's immune response. HLA-G is also pathologically expressed in a diverse set of tumors, acting as an immunescape mechanism. Trough splicing mechanisms, HLA-G can be expressed as a membrane-bound or as a soluble isoform. Interestingly, the transference of membrane fragments between cells, a process called trogocytosis, can spread HLA-G inhibitory function beyond the reach of HLA-G-expressing cells. For instance, upon trogocytosis, effector CD4+ T cells stop proliferating, stop responding to stimulation, and behave as regulatory T cells. Recently, mesenchymal stromal cells (MSC) were shown to secrete soluble HLA-G, adding to the existing set of secreted molecules, by which MSC can modulate cells of the immune system. Despite the importance of secreted factors, cell-to-cell contacts have an important role in the immunological effects exerted by MSC. Based in these facts, we hypothesized that one of the mechanisms used by MSC to immunomodulate T cells, could involve trogocytosis mediated HLA-G transference. To test this, CD3+ T cell were immunomagnetically selected from peripheral blood mononuclear cells (PBMC) and pre-activated for 72hs using anti-CD2/CD3/CD28 beads. Activated T-cells were then incubated for 30 min, alone or with MSC. After this period, cells were recovered and the transfer of membrane bound molecules from MSC to T-cells was analyzed by flow cytometry (3 experiments) using antibodies against HLA-G. Confocal microscopy was carried in an additional experiment, using antibodies against HLA-G, CD140B (a specific MSC marker) and CD3. DAPI was used for nuclear staining. Flow cytometry analysis revealed that activated CD3+ T-cells did not express HLA-G, while MSC expressed HLA-G intracytoplasmatic. After 30 min of co-incubation, membrane bound HLA-G was detected in a variable percentage of CD3+ cells, ranging from 3 to up to 16%, characterizing the transfer of HLA-G from MSC to CD3+ T-cells. Confocal microscopy revealed that activated T-cells cultured alone did not stained for HLA-G or CD140B, while, about 12% of the CD3+ cells stained for membrane HLA-G, following the 30 min incubation with MSC. Additionally, CD140B was also transferred from MSC to CD3+ T-cells. These data are the first demonstration of trogocytosis mediated transfer of HLA-G from MSC to activated T lymphocytes. Given the significance of HLA-G expression and trogocytosis in normal and pathological situations, this newly described immunological mechanism should be considered in the development and application of MSC-based therapies. Supported by CNPq and FAPESP. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Catastrophic NAD+ Depletion in Activated T Lymphocytes through Nampt Inhibition Reduces Demyelination and Disability in EAE.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4732-4732
Author(s):  
Santina Bruzzone ◽  
Floriana Fruscione ◽  
Sara Morando ◽  
Alessandro Poggi ◽  
Anna Garuti ◽  
...  
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
T Lymphocyte ◽  
Conflicts Of Interest ◽  
Lymphocyte Function ◽  
Activation Markers ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Nad Synthesis ◽  
Activated T Lymphocytes ◽  
Nad Depletion

Abstract Abstract 4732 Nicotinamide phosphoribosyltransferase (Nampt) inhibitors such as FK866 are potent inhibitors of NAD+ synthesis that show promise for the treatment of different forms of cancer. Based on Nampt upregulation in activated T lymphocytes and on preliminary reports of lymphopenia in FK866 treated patients, we have investigated FK866 for its capacity to interfere with T lymphocyte function and survival. Intracellular pyridine nucleotides, ATP, mitochondrial function, viability, proliferation, activation markers and cytokine secretion were assessed in resting and in activated human T lymphocytes. In addition, we used experimental autoimmune encephalomyelitis (EAE) as a model of T-cell mediated autoimmune disease to assess FK866 efficacy in vivo. We show that activated, but not resting, T lymphocytes undergo massive NAD+ depletion upon FK866 mediated Nampt inhibition. As a consequence, impaired proliferation, reduced TNF-a production, ATP shortage, and, finally, autophagic cell demise result. We demonstrate that upregulation of the of the NAD+-degrading enzyme poly-(ADP-ribose)-polymerase (PARP) by activated T cells enhances their susceptibility to NAD+ depletion. We relate defective TNF-a production in response to FK866 to impaired SIRT6 activity. Finally, in line with its capacity to interfere with T lymphocyte function and survival, we show that FK866 strikingly reduces the neurological damage and the clinical manifestations of EAE. In conclusion, Nampt inhibitors (and possibly sirtuin inhibitors) could be used to modulate T cell-mediated immune responses and thereby be beneficial in immune-mediated disorders. Disclosures: No relevant conflicts of interest to declare.

scholarly journals POS0369 ELEVATED EXPRESSION OF TIM-3 ON NEUTROPHILS CORRELATES WITH DISEASE ACTIVITY AND SEVERITY OF ANKYLOSING SPONDYLITIS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 414.2-415
Author(s):  
X. Huang ◽  
T. W. Li ◽  
J. Chen ◽  
Z. Huang ◽  
S. Chen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Ankylosing Spondylitis ◽  
Disease Activity ◽  
Immune Cells ◽  
Clinical Manifestations ◽  
Innate Immune ◽  
Innate Immune Cells ◽  
Bacterial Killing ◽  
Markers Of Inflammation ◽  
Significant Difference

Background:Ankylosing spondylitis (AS) is a type of common, chronic inflammatory disease that compromises the axial skeleton and sacroiliac joints, causing inflammatory low back pain and progressive spinal stiffness, over time some patients develop spinal immobility and ankylosis which can lead to a decrease in quality of life. The last few decades, evidence has clearly indicated that neutrophil also plays key roles in the progression of AS. However, the immunomodulatory roles and mechanisms of neutrophils in AS are poorly understood. T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) has been reported as an important regulatory molecule, expressed and regulated on different innate immune cells, plays a pivotal role in several autoimmunity diseases. Recent study indicates that Tim3 is also expressed on neutrophils. However, the frequency and roles of Tim3-expressing neutrophils in AS was not clear.Objectives:In this study, we investigated the expression of Tim3 on neutrophils in AS patients and explored the correlation between the level of Tim3-expressing neutrophils and the disease activity and severity of AS.Methods:Patients with AS were recruited from Guangdong Second Provincial General Hospital (n=62). Age/sex-matched volunteers as Healthy controls (HC) (n=39). The medical history, clinical manifestations, physical examination, laboratory measurements were recorded. The expression of costimulatory molecules including programmed death 1 (PD-1), Tim-3 on neutrophils were determined by flow cytometry. The mRNA expression of PD-1 and Tim-3 was determined by real-time PCR. The levels of Tim3-expressing neutrophils in AS patients were further analyzed for their correlation with the markers of inflammation such as ESR,CRP,WBC and neutrophil count(NE), as well as disease activity and severity of AS. The expression of Tim3 on neutrophils was monitored during the course of treatment (4 weeks).Results:The expression of Tim3 on neutrophils in patients with AS was increased compared to the HC (Figure 1A). However, significant difference was observed in the frequency of PD-1-expressing neutrophils between AS patients and HC (Figure 1B). The expression analysis of Tim-3 mRNA, but not PD-1, confirmed the results obtained from flow cytometry (Figure 1C). The level of Tim3-expressing neutrophils in patients with AS showed an positive correlation with ESR, CRP and ASAS-endorsed disease activity score (ASDAS) (Figure 1D). Moreover, the frequency of Tim3-expressing neutrophils in active patients(ASDAS≥1.3) was increased as compare with the inactive patients (ASDAS<1.3) (Figure 1E). As shown in Figure 1F, the frequency of Tim3-expressing neutrophils decreased after the treatment.Conclusion:Increased Tim-3 expression on neutrophils may be a novel indicator to assess disease activity and severity in AS, which may serves as a negative feedback mechanism preventing potential tissue damage caused by excessive inflammatory responses in AS patients.References:[1]Han, G., Chen, G., Shen, B. & Li, Y., Tim-3: an activation marker and activation limiter of innate immune cells. FRONT IMMUNOL 4 449 (2013).[2]Vega-Carrascal, I. et al., Galectin-9 signaling through TIM-3 is involved in neutrophil-mediated Gram-negative bacterial killing: an effect abrogated within the cystic fibrosis lung. J IMMUNOL 192 2418 (2014).Figure 1.(A,B)The expression of Tim3 and PD-1 on neutrophils in AS and HC were determined by flow cytometry.(C) The expression of Tim3 and PD-1 on neutrophils in AS and HC were determined by RT-PCR.(D)The correction between Tim3-expressing neutrophils and ESR,CRP,ASDAS.(E) The expression of Tim3 on neutrophils in active and inactive patients.(F) Influence of treatment on the frequency of Tim3-expressing neutrophils.Disclosure of Interests:None declared

scholarly journals FRI0015 PHENOTYPE AND FUNCTION OF THE PERIPHERAL BLOOD DENDRITIC CELLS OF PSORIASIS PATIENTS WITH AND WITHOUT ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 578.1-579
Author(s):  
S. Schnitte ◽  
A. Fuchs ◽  
T. Funk ◽  
A. C. Pecher ◽  
D. Dörfel ◽  
...  
Keyword(s):  
Risk Factors ◽  
Flow Cytometry ◽  
Dendritic Cells ◽  
Psoriatic Arthritis ◽  
T Lymphocytes ◽  
Peripheral Blood ◽  
Surface Proteins ◽  
Research Support ◽  
Cross Sectional ◽  
Cell Counts

Background:Psoriasis is a frequent skin disease that can appear with an arthritic manifestation in approximately 30% of the cases [1]. The underlying excessive immune reaction caused by pro-inflammatory cytokines can be triggered by several risk factors [2]. Various subgroups of Dendritic cells (DCs) in the skin play a crucial role in the induction of the dermal inflammatory response [3].Objectives:As the role of peripheral blood DCs remains unknown and the cause of an arthritic manifestation is still not completely understood [4], this project aimed to detect differences in phenotype or function of peripheral blood DCs in psoriatic patients with or without arthritis.Methods:We analyzed peripheral blood cells of 60 psoriasis patients with and without arthritis. Different DC subpopulations were detected by flow cytometry. Monocyte-derived DCs were cultured with or without Lipopolysaccharides to gain immature (iDC) and mature (mDC) cells. The DC phenotype was determined by staining with CD80, CD83, CD86, CD206, CCR7, CD1a, HLA-DR, CD40, GPN-MB, DC209 and CD14. Their T-cell stimulatory capability was analyzed by co-incubation with Carboxyfluorescein succinimidyl ester stained lymphocytes and the quantification of CD4+ T-lymphocytes afterwards. To measure the migration capacity DCs were seated into transwell chambers with a semipermeable membrane and partly supplemented with Macrophage Inflammatory Protein 3 Beta (Mip3b). Migrated cells were detected by flow cytometry. Measured cell counts were normalized to cell counts without Mip3b stimulation.Results:Comparing the factor of increase of migrated mDC counts due to mip3b stimulation, we detected a significant lower rate in samples of patients with arthritis (PsA) compared to those of patients without (Ps). Assays of mDCs without mip3b stimulation showed a significant higher count of migrated cells in the samples of the arthritic group [Figure 1]. Cell counts with Mip3b stimulation did vary slightly in the groups. The DC subpopulations and the expression of analyzed cell surface proteins did not show significant differences. The amounts of stimulated T-Lymphocytes did not differ significantly.Figure 1.Migration essay showing mDCs following Mip3b (+miß3b) as multiples of mDCs without stimulation (-mip3b). The factor of increase is significantly lower in patients with arthritis (PsA) compared to patients without (Ps). Absolute counts of migrated mDCs without Mip3b are significantly higher in the arthritic group. Cell counts with stimulation do not differ significantly (data not shown). N=24, p<0.05Conclusion:CCL19 (Mip3b) is a potent ligand to the CCR7 receptor inducing migration of DCs towards the lymphatic node [5]. The CCR7 amounts on the DC surface did not differ significantly in the groups. The mDCs without CCL19 stimulation migrated in higher amounts in samples of arthritic patients. Cell counts of stimulated DCs showed only slight differences. These results could be generated by a different appearance of the DCs of arthritic patients that might facilitate migration. Further experiments focusing on this aspect should be performed. A possible effect of disruptive factors (age, sex, medication…) needs to be clarified.References:[1]Henes, J.C., et al.,High prevalence of psoriatic arthritis in dermatological patients with psoriasis: a cross-sectional study.Rheumatol Int, 2014.34(2): p. 227-34.[2]Lee, E.B., et al.,Psoriasis risk factors and triggers.Cutis, 2018.102(5s): p. 18-20.[3]Kim, T.G., S.H. Kim, and M.G. Lee,The Origin of Skin Dendritic Cell Network and Its Role in Psoriasis.Int J Mol Sci, 2017.19(1).[4]Veale, D.J. and U. Fearon,The pathogenesis of psoriatic arthritis.Lancet, 2018.391(10136): p. 2273-2284.[5]Ricart, B.G., et al.,Dendritic cells distinguish individual chemokine signals through CCR7 and CXCR4.J Immunol, 2011.186(1): p. 53-61.Acknowledgments:This project was financially supported by Novartis Pharma GmbH.Disclosure of Interests:Sarah Schnitte Grant/research support from: Reaserch grant by Novartis, Alexander Fuchs: None declared, Tanja Funk: None declared, Ann-Christin Pecher: None declared, Daniela Dörfel: None declared, Jörg Henes Grant/research support from: Novartis, Roche-Chugai, Consultant of: Novartis, Roche, Celgene, Pfizer, Abbvie, Sanofi, Boehringer-Ingelheim,

Immunophenotyping of T Lymphocytes by Three-Color Flow Cytometry in Healthy Newborns, Children, and Adults

1997 ◽  
Vol 84 (1) ◽  
pp. 46-55 ◽  
Author(s):  
Thomas W. McCloskey ◽  
Terri Cavaliere ◽  
Saroj Bakshi ◽  
Rita Harper ◽  
James Fagin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Lymphocytes ◽  
Color Flow
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