Flow cytometry evaluation of the rhesus monkey cellular immunity following the Zaire ebolavirus (Filoviridae; Ebolavirus: Zaire ebolavirus) experimental infection

Introduction. The outbreaks of the Zaire ebolavirus (ZE) disease (ZED) that have arisen in the last decade determine the need to study the infection pathogenesis, the formation of specific immunity forming as well as the development of effective preventive and therapeutic means. All stages of fight against the ZED spread require the experimental infection in sensitive laboratory animals, which are rhesus monkeys in case of this disease .The aim of the study is to evaluate the rhesus monkey cellular immunity following the ZE experimental infection by the means of flow cytometry (cytofluorimetry).Material and methods. Male rhesus monkeys were intramuscularly infected by the dose of 15 LD50 (dose of the pathogen that causes 50% mortality of infected animals) of the ZE, the Zaire strain (ZEBOV). Levels of 18 peripheral blood lymphocyte populations of the animals before the ZE experimental infection and at the terminal stage of the disease were assessed using flow cytometry.Results and discussion. The certain changes in the levels of the lymphocyte populations were observed following infection, indicating simultaneous activation and suppression of the immune system during ZED. The increase in content was observed for T-lymphocytes, T-helper and cytotoxic T-lymphocytes expressing the corresponding markers of early activation. The decrease was recorded for T-lymphocytes and double-positive T-lymphocytes expressing corresponding markers of late activation, as well as natural killer cells expressing CD8 (p < 0.05).Conclusion. For the first time in the Russian Federation, the rhesus monkey cellular immunity before and after the ZE experimental infection was assessed using flow cytometry.

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Expression of CD25highRegulatory T Cells and PD-1 in Gastric Infiltrating CD4+T Lymphocytes in Patients with Helicobacter pylori Infection

Clinical and Vaccine Immunology ◽

10.1128/cvi.00422-10 ◽

2011 ◽

Vol 18 (7) ◽

pp. 1198-1201 ◽

Cited By ~ 14

Author(s):

Yi-Ying Wu ◽

Jiunn-Horng Chen ◽

Jung-Ta Kao ◽

Kuo-Ching Liu ◽

Chih-Ho Lai ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Helicobacter Pylori ◽

T Lymphocytes ◽

Cellular Immunity ◽

Cd4 T Cells ◽

Helicobacter Pylori Infection ◽

Content Type

ABSTRACTWe observed by flow cytometry that the frequency of both gastric infiltrating Tregs and PD-1-positive CD4 T cells is correlated with the density ofHelicobacter pylori, suggesting that cellular immunity against this pathogen is inhibited.

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Flow cytometric detection of receptors for interleukin-6 on bone marrow and peripheral blood cells of humans and rhesus monkeys

Blood ◽

10.1182/blood.v81.8.2036.bloodjournal8182036 ◽

1993 ◽

Vol 81 (8) ◽

pp. 2036-2043

Author(s):

AW Wognum ◽

FC van Gils ◽

G Wagemaker

Keyword(s):

Bone Marrow ◽

T Lymphocytes ◽

Rhesus Monkey ◽

Interleukin 6 ◽

Peripheral Blood ◽

Blood Cells ◽

Rhesus Monkeys ◽

Cell Types ◽

Cd8 T Lymphocytes ◽

Blast Cells

The expression of receptors for interleukin-6 (IL-6) on human and rhesus monkey peripheral blood and bone marrow (BM) cells was examined by multiparameter flow cytometry after staining with biologically active, biotin-labeled human IL-6 and phycoerythrin-conjugated streptavidin. Consistent with the multiple biologic effects of IL-6 in stimulating immune functions and hematopoiesis, IL-6 receptors were detectable on a wide variety of cell types. In peripheral blood, IL-6 receptors were detectable on monocytes, granulocytes, and on CD4+ T lymphocytes but not on resting, CD19+ B lymphocytes and CD56+ natural killer (NK) cells. CD8+ T lymphocytes also expressed IL-6 receptors but at lower levels than CD4+ cells. The IL-6 receptors on granulocytes were only detectable after staining with high concentrations of biotin- IL-6, suggesting that most IL-6 receptors on these cells represent low- affinity sites. In contrast, IL-6 receptors on both CD4+ and CD8+ T lymphocytes were detectable at biotin-IL-6 concentrations as low as 10 pmol/L, indicating that these cells bind IL-6 with high affinity. IL-6 receptor expression patterns on rhesus monkey and human blood cells were very similar except that receptor levels on granulocytes were lower in humans than in rhesus monkeys. Similar differences in expression levels were observed for IL-6 receptors that were detectable on most granulocyte precursors in the mononuclear fraction of rhesus monkey and human bone marrow. In addition to these relatively mature cell types, IL-6 receptors were detectable on a large fraction of human and rhesus monkey BM blast cells that express the CD34 antigen. The presence of IL-6 receptors on CD34+ BM blast cells, which are the precursor cells of most, if not all, BM-derived blood cells, is consistent with the ability of IL-6, in conjunction with other cytokines, to stimulate immature hemopoietic cells in vitro and to promote blood cell production when administered in vivo.

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A Commonly Recognized Simian Immunodeficiency Virus Nef Epitope Presented to Cytotoxic T Lymphocytes of Indian-Origin Rhesus Monkeys by the Prevalent Major Histocompatibility Complex Class I Allele Mamu-A*02

Journal of Virology ◽

10.1128/jvi.75.21.10179-10186.2001 ◽

2001 ◽

Vol 75 (21) ◽

pp. 10179-10186 ◽

Cited By ~ 24

Author(s):

Suzanne Robinson ◽

William A. Charini ◽

Michael H. Newberg ◽

Marcelo J. Kuroda ◽

Carol I. Lord ◽

...

Keyword(s):

T Lymphocytes ◽

Rhesus Monkey ◽

Simian Immunodeficiency Virus ◽

Mhc Class I ◽

T Lymphocyte ◽

Rhesus Monkeys ◽

Cytotoxic T Lymphocyte ◽

Class I ◽

Mhc Class I Molecule ◽

Immunodeficiency Virus

ABSTRACT The ability to monitor vaccine-elicited CD8+ cytotoxic T-lymphocyte (CTL) responses in simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys has been limited by our knowledge of viral epitopes predictably presented to those lymphocytes by common rhesus monkey MHC class I alleles. We now define an SIV and SHIV Nef CTL epitope (YTSGPGIRY) that is presented to CD8+ T lymphocytes by the common rhesus monkey MHC class I molecule Mamu-A*02. All seven infectedMamu-A*02 + monkeys evaluated demonstrated this response, and peptide-stimulated interferon gamma Elispot assays indicated that the response represents a large proportion of the entire CD8+ T-lymphocyte SIV- or SHIV-specific immune response of these animals. Knowledge of this epitope and MHC class I allele substantially increases the number of available rhesus monkeys that can be used for testing prototype HIV vaccines in this important animal model.

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Flow cytometric detection of receptors for interleukin-6 on bone marrow and peripheral blood cells of humans and rhesus monkeys

Blood ◽

10.1182/blood.v81.8.2036.2036 ◽

1993 ◽

Vol 81 (8) ◽

pp. 2036-2043 ◽

Cited By ~ 38

Author(s):

AW Wognum ◽

FC van Gils ◽

G Wagemaker

Keyword(s):

Bone Marrow ◽

T Lymphocytes ◽

Rhesus Monkey ◽

Interleukin 6 ◽

Peripheral Blood ◽

Blood Cells ◽

Rhesus Monkeys ◽

Cell Types ◽

Cd8 T Lymphocytes ◽

Blast Cells

Abstract The expression of receptors for interleukin-6 (IL-6) on human and rhesus monkey peripheral blood and bone marrow (BM) cells was examined by multiparameter flow cytometry after staining with biologically active, biotin-labeled human IL-6 and phycoerythrin-conjugated streptavidin. Consistent with the multiple biologic effects of IL-6 in stimulating immune functions and hematopoiesis, IL-6 receptors were detectable on a wide variety of cell types. In peripheral blood, IL-6 receptors were detectable on monocytes, granulocytes, and on CD4+ T lymphocytes but not on resting, CD19+ B lymphocytes and CD56+ natural killer (NK) cells. CD8+ T lymphocytes also expressed IL-6 receptors but at lower levels than CD4+ cells. The IL-6 receptors on granulocytes were only detectable after staining with high concentrations of biotin- IL-6, suggesting that most IL-6 receptors on these cells represent low- affinity sites. In contrast, IL-6 receptors on both CD4+ and CD8+ T lymphocytes were detectable at biotin-IL-6 concentrations as low as 10 pmol/L, indicating that these cells bind IL-6 with high affinity. IL-6 receptor expression patterns on rhesus monkey and human blood cells were very similar except that receptor levels on granulocytes were lower in humans than in rhesus monkeys. Similar differences in expression levels were observed for IL-6 receptors that were detectable on most granulocyte precursors in the mononuclear fraction of rhesus monkey and human bone marrow. In addition to these relatively mature cell types, IL-6 receptors were detectable on a large fraction of human and rhesus monkey BM blast cells that express the CD34 antigen. The presence of IL-6 receptors on CD34+ BM blast cells, which are the precursor cells of most, if not all, BM-derived blood cells, is consistent with the ability of IL-6, in conjunction with other cytokines, to stimulate immature hemopoietic cells in vitro and to promote blood cell production when administered in vivo.

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Immunohistochemical Study of Lymphocyte Populations Infiltrating the Gastric Mucosa of Beagle Dogs Experimentally Infected withHelicobacter pylori

Infection and Immunity ◽

10.1128/iai.68.8.4769-4772.2000 ◽

2000 ◽

Vol 68 (8) ◽

pp. 4769-4772 ◽

Cited By ~ 23

Author(s):

Giacomo Rossi ◽

Damiano Fortuna ◽

Laura Pancotto ◽

Giacomo Renzoni ◽

Ennio Taccini ◽

...

Keyword(s):

Helicobacter Pylori ◽

T Lymphocytes ◽

Gastric Mucosa ◽

B Lymphocytes ◽

Experimental Infection ◽

Mononuclear Cells ◽

Immunohistochemical Study ◽

Beagle Dogs ◽

Lymphoid Follicles ◽

Lymphocyte Populations

ABSTRACT Experimental infection of beagle dogs with Helicobacter pylori induces recruitment to the gastric mucosae of neutrophils at early stages and later of mononuclear cells that organize into lymphoid follicles. These structures become macroscopically evident and consist of peripheral CD4+ T lymphocytes and central CD21+ B lymphocytes. Furthermore, transient expression of interleukin-8 (IL-8) parallels the presence of neutrophils in the gastric mucosae, whereas expression of IL-6 tends to persist chronically.

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Ex Vivo IL-2 Treatment Increases the Immunosuppressive Subset of T Lymphocytes to Suppress Allo-Immune Response

Blood ◽

10.1182/blood-2021-152462 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2066-2066

Author(s):

Jeong-A Kim ◽

Jinhang KIM ◽

Misuk Yang ◽

Youngrok Park ◽

Jae-Yong Kwak

Keyword(s):

Flow Cytometry ◽

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Lymphocytes ◽

Cell Sorting ◽

Peripheral Blood ◽

Low Dose ◽

T Cell Proliferation ◽

Double Positive

Abstract T lymphocytes are critical players in graft-versus-host disease (GVHD); however, their specific roles and mechanisms in GVHD have not been clearly defined. Co-inhibitory receptors including PD1, TIM3, TIGIT, and LAG3 play a key role in regulating T cell responses and maintaining immune homeostasis. TIM3 expression on lymphocytes was identified to induce immune tolerance in a mouse GVHD model; in addition, PD1 +TIM3 + double-positive T cells displayed more potent immunosuppression compared to PD1 + T cells. However, despite the strong immunosuppression exerted by PD1 +TIM3 + double-positive T cells, their clinical potential is greatly limited by their low cell number in peripheral blood. In this study, we introduce a novel method to isolate CD3 + cells from G-CSF mobilized peripheral blood stem cells (G-PBSCs), and culture CD3 +PD1 +TIM3 + lymphocytes to treat GVHD. Methods. The donors were subcutaneously injected with G-CSF (10μg/kg) for five days. G-PBSCs were collected from the donors using a COBE spectra cell separator. Then, the highly purified CD3 + cells were isolated by positive selection with magnetic-activated cell sorting (MACS) from G-PBSCs using CD3 Dynabeads™. Isolated CD3 + cells were cultured with a low concentration of IL-2 (50U/mL), and PD1, TIM3, LAG3, and TIGIT expressions were assessed using flow cytometry (FACS). CD3 +PD1 +TIM3 +, CD3 +PD1 +TIM3 +LAG3 +TIGIT - and CD3 +PD1 +TIM3 +LAG3 +TIGIT +cells are sorted and cultured with irradiated allo-MNCs for 4 days (Mixed Lymphocyte Reaction; MLR). We used 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) as an intracellular fluorescent dye in MLR (CFSE-MLR) to measure of T cells proliferation. Results. In normal, untreated G-PBSCs, very low percentages of cells are CD3 +PD1 +TIM3 +, CD3 +PD1 +LAG3 + and CD3 +PD1 +TIGIT + (0.6±0.4, 0.3±0.2 and 1.3±0.5% in G-PBSC, respectively). However, after treating G-PBSC cells with a low concentration of IL-2 (50 U/mL), we discovered the percentages of CD3 +PD1 +TIM3 +, CD3 +PD1 +LAG3 +, and CD3 +PD1 +TIGT + lymphocytes in G-PBSCs markedly increased by 19.6±5.9, 18.5±4.3, 17.7±6.5%, respectively. Of note, there was a very small change (~1.2 fold increase) in the total number of CD3 + lymphocytes, which indicates that the low dose IL-2 therapy alters subpopulations of T lymphocytes, rather than increasing the proliferation rate. Next, we tested the immunosuppressive capacity of the cultured CD3 +PD1 +TIM3 +cells. To do this, we used CSFE-MLR to measure T cell proliferation. CD3 +PD1 +TIM3 + lymphocytes were depleted in CD3 + G-PBSCs by flow cytometry-based cell sorting. Then we cultured the CD3 + G-PBSCs with allo-MNCs and discovered that the CD3 + G-PBSCs that lacked CD3 +PD1 +TIM3 + lymphocytes demonstrated a significantly increased level of T-cell proliferation compared to CD3 + G-PBSCs, and thereby confirming the immunosuppressive function of CD3 +PD1 +TIM3 + lymphocytes in inhibiting T cell proliferation (% of unstimulated T cells in CD3 + G-PBSCs vs. in CD3 +PD1 +TIM3 + depleted G-PBSC CD3 + cells; 33.5±7.5% vs. 6.3±4.2%). After confirming the immunosuppressive function of the IL-2 treated CD3 +PD1 +TIM3 + lymphocytes, we investigated expressions of other co-inhibitory surface markers such as LAG3 and TIGIT. Amongst the cultured CD3 +PD1 +TIM3 + lymphocytes, 96.2±3.3% of the CD3 +PD1 +TIM3 + lymphocytes were LAG3 +, 28.9±5.4% of the CD3 +PD1 +TIM3 + lymphocytes were TIGIT +, and 63.7±7.8% of the cells were TIGIT -. Interestingly, CD3 +PD1 +TIM3 +LAG3 +TIGIT - lymphocytes demonstrated significantly enhanced levels of immunosuppression compared to CD3 +PD1 +TIM3 +LAG3 +TIGIT + lymphocytes (% of unstimulated T cells in CD3 +PD1 +TIM3 +LAG3 +TIGIT - lymphocytes vs. in CD3 +PD1 +TIM3 +LAG3 +TIGIT + lymphocytes, 53.8±5.5% vs. 23.2±7.4%). Conclusion. We demonstrate that treating CD3 + G-PBSCs with low-dose IL-2 markedly increases the percentage of CD3 +PD1 +TIM3 + lymphocytes, which is known to exert strong immunosuppression. Also, we suggest that CD3 +PD1 +TIM3 +LAG3 +TIGIT - lymphocytes are the subpopulation within CD3 +PD1 +TIM3 + cells that demonstrate the most potent immunosuppression compared to other subpopulations of CD3 +PD1 +TIM3 + lymphocytes. Taken together, such findings suggest that the low-dose IL-2 therapy has therapeutic potential to treat patients with GVHD. Disclosures No relevant conflicts of interest to declare.

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The Growth of Herpesvirus mulatto in Human Fibroblast

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051232 ◽

1974 ◽

Vol 32 ◽

pp. 244-245

Author(s):

Glennelle Washington ◽

Philip P. McGrath ◽

Peter R. Graze ◽

Ivor Royston

Keyword(s):

Rhesus Monkey ◽

Microscopic Study ◽

Electron Microscopic Study ◽

Peripheral Blood ◽

Human Fibroblast ◽

Rhesus Monkeys ◽

Human Fibroblasts ◽

Electron Microscopic ◽

Specific Antisera ◽

Peripheral Blood Leucocytes

Herpes-like viruses were isolated from rhesus monkey peripheral blood leucocytes when co-cultivated with WI-38 cells. The virus was originally designated rhesus leucocyte-associated herpesvirus (LAHV) and subsequently called Herpesvirus mulatta (HVM). The original isolations were from juvenile rhesus monkeys shown to be free of antibody to rhesus cytomegalic virus. The virus could only be propagated in human or simian fibroblasts. Use of specific antisera developed from HVM showed no relationship between this virus and other herpesviruses. An electron microscopic study was undertaken to determine the morphology of Herpesvirus mulatta (HVM) in infected human fibroblasts.

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Intratracheal inoculation of human varicella zoster virus (VZV; MAV strain) vaccine successfully induced VZV IgG antibodies in rhesus monkeys

Laboratory Animal Research ◽

10.1186/s42826-021-00091-3 ◽

2021 ◽

Vol 37 (1) ◽

Author(s):

Jong-Min Kim ◽

Chung-Gyu Park

Keyword(s):

Rhesus Monkey ◽

Varicella Zoster Virus ◽

Humoral Immunity ◽

Antibody Production ◽

Rhesus Monkeys ◽

Igg Antibodies ◽

Simian Varicella Virus ◽

Varicella Zoster ◽

Varicella Virus ◽

Strain Vaccine

Abstract Background The objective of this study was to investigate whether the use of live attenuated varicella zoster virus (VZV) MAV vaccination can efficiently induce VZV antibody production in naive rhesus monkeys as an approach to prevent simian varicella virus (SVV) reactivation in animals immunosuppressed for transplantation studies. Results Clinically available human VZV vaccine was used to induce the production of anti-VZV antibodies in rhesus monkeys. A vial of the vaccine was subcutaneously injected at 0 week, and the second and third vaccination was performed at 5 and 6 weeks by intratracheal inoculation. The titer of anti-VZV IgG was assessed at 0, 2, 4, 6, and 7 weeks. At 2 weeks, 3/16 were seropositive for VZV IgG. At 6 weeks, 9/16 were shown to be seropositive. At 7 weeks, 16/16 were found to be seropositive. Conclusions The VZV vaccine via intratrachael inoculation was shown to induce VZV IgG humoral immunity in rhesus monkeys and may be important immunosuppressed macaques for transplantation studies. Although the humoral immunity produced is an important finding, further studies will be necessary to confirm possible protection and it could protect probably against SVV infection in rhesus monkey.

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

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.4296 ◽

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,

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