scholarly journals Immune profiling of influenza‐specific B‐ and T‐cell responses in macaques using flow cytometry‐based assays

2020 ◽  
Vol 99 (1) ◽  
pp. 97-106 ◽  
Author(s):  
Marios Koutsakos ◽  
Toshiki Sekiya ◽  
Brendon Y Chua ◽  
Thi Hoang Oanh Nguyen ◽  
Adam K Wheatley ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
T Cell Responses ◽  
Cell Responses ◽  
Immune Profiling

scholarly journals T cell mediated immunity against influenza H5N1 nucleoprotein, matrix and hemagglutinin derived epitopes in H5N1 survivors and non-H5N1 subjects

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11021
Author(s):  
Pirom Noisumdaeng ◽  
Thaneeya Roytrakul ◽  
Jarunee Prasertsopon ◽  
Phisanu Pooruk ◽  
Hatairat Lerdsamran ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Influenza Virus ◽  
T Cell ◽  
Avian Influenza ◽  
Influenza Vaccine ◽  
Elispot Assay ◽  
H5n1 Virus ◽  
T Cell Responses ◽  
Cell Responses ◽  
Influenza H5n1

Background Protection against the influenza virus by a specific antibody is relatively strain specific; meanwhile broader immunity may be conferred by cell-mediated immune response to the conserved epitopes across influenza virus subtypes. A universal broad-spectrum influenza vaccine which confronts not only seasonal influenza virus, but also avian influenza H5N1 virus is promising. Methods This study determined the specific and cross-reactive T cell responses against the highly pathogenic avian influenza A (H5N1) virus in four survivors and 33 non-H5N1 subjects including 10 H3N2 patients and 23 healthy individuals. Ex vivo IFN-γ ELISpot assay using overlapping peptides spanning the entire nucleoprotein (NP), matrix (M) and hemagglutinin (HA) derived from A/Thailand/1(KAN-1)/2004 (H5N1) virus was employed in adjunct with flow cytometry for determining T cell functions. Microneutralization (microNT) assay was performed to determine the status of previous H5N1 virus infection. Results IFN-γ ELISpot assay demonstrated that survivors nos. 1 and 2 had markedly higher T cell responses against H5N1 NP, M and HA epitopes than survivors nos. 3 and 4; and the magnitude of T cell responses against NP were higher than that of M and HA. Durability of the immunoreactivity persisted for as long as four years after disease onset. Upon stimulation by NP in IFN-γ ELISpot assay, 60% of H3N2 patients and 39% of healthy subjects exhibited a cross-reactive T cell response. The higher frequency and magnitude of responses in H3N2 patients may be due to blood collection at the convalescent phase of the patients. In H5N1 survivors, the effector peptide-specific T cells generated from bulk culture PBMCs by in vitro stimulation displayed a polyfunction by simultaneously producing IFN-γ and TNF-α, together with upregulation of CD107a in recognition of the target cells pulsed with peptide or infected with rVac-NP virus as investigated by flow cytometry. Conclusions This study provides an insight into the better understanding on the homosubtypic and heterosubtypic T cell-mediated immune responses in H5N1 survivors and non-H5N1 subjects. NP is an immunodominant target of cross-recognition owing to its high conservancy. Therefore, the development of vaccine targeting the conserved NP may be a novel strategy for influenza vaccine design.

Immunomagnetic Depletion of CD25-Expressing T Cells from Donor Blood Removes a Mixed Population of Regulatory and Effector T Cells - Implications for Graft Engineering.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 724-724
Author(s):  
J. Joseph Melenhorst ◽  
Jun Lu ◽  
Edgardo Sosa ◽  
Nancy F. Hensel ◽  
A. John Barrett
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
T Cell Responses ◽  
Substantial Proportion ◽  
Absolute Increase ◽  
Cell Responses ◽  
Treg Population

Abstract CD4+CD25+FOXP3+ regulatory T cells (TR) control proliferative CD4 and CD8 T cell responses to self and foreign antigens such as cytomegalovirus (CMV) and tumor-specific antigens. Thus, depletion of CD25-expressing cells from a resting population of T cells prior to antigen stimulation could boost the generation of antigen-specific T cells for adoptive transfer to treat viral infection or tumors. We depleted peripheral blood mononuclear cells (PBMC) from nine CMV seropositive donors using Miltenyi CD25 microbeads (20 μl/107 cells). CD25-depleted or unmanipulated PBMC were stimulated with CMV pp65-expressing antigen presenting cells for 10–14 days with low dose IL-2, and intracellular interferon-gamma (IFNγ) production by flow cytometry was compared between CD25-depleted and -undepleted cultures. An absolute increase in antigen-specific CD4+ and CD8+ T cells was seen after CD25 depletion in 4/8 and 5/8 cultures respectively. However, in other cultures there was a decrease or no change in IFNγ+ CD4+ T cells in CD25-depleted cultures, suggesting that the pp65-specific precursor cells had also been removed. We then used 4 μl beads per 107 PBMC to selectively remove only the CD25bright (predominantly Treg) population in nine donors and confirmed by flow cytometry that only CD25bright cells had been removed from the starting population. However, real-time quantitative PCR (Q-PCR) showed that even though the CD25+ fraction was enriched in FOXP3-expressing cells, a substantial proportion of the CD25-depleted PBMC still expressed FOXP3. Flow cytometric analysis of FOXP3 expression by CD25+ and CD25-negative CD4+ T cells showed that a substantial proportion of CD25- cells expressed FOXP3, confirming that CD25 is not a suitable single marker for depletion of Tregs. Again no reproducible augmentation of antigen-specific T cell responses was observed: one and five donors showing an increase in CD4 and CD8+ antigen-specific T cells, respectively, while the remainder showed a decrease or no change in CD4+ and CD8+ IFNγ-producing cells. These results suggest that removal of CD25+ cells from PBMC using CD25 microbeads removes both Treg and pp65-specific effector CD4+ and CD8+ T cells. Further, since FOXP3 is induced in responder cells as confirmed by FOXP3 Q-PCR, depletion of Treg at the start of the cultures may only transiently alleviate the negative regulation of the antigen response. Thus, CD25 depletion using microbeads is not a reliable method to boost antigen specific T cell expansions because of the inadvertent removal of a portion of the memory response to the antigen. Since it recently has been demonstrated that Treg act as an IL-2 sink, the addition of this cytokine should functionally silence the Tregs while preserving the inflammatory response.

scholarly journals Strong anti-viral responses in pediatric COVID-19 patients in South Brazil

2021 ◽  
Author(s):  
Tiago Fazolo ◽  
Karina Lima ◽  
Julia C. Fontoura ◽  
Priscila Oliveira de Souza ◽  
Gabriel Hilario ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
T Cell Responses ◽  
Humoral Immune ◽  
Cell Responses ◽  
Humoral Immune Responses ◽  
Hla Dr ◽  
Data Points ◽  
Viral Responses ◽  
Immune Profiling

AbstractEpidemiological evidence that COVID-19 manifests as a milder disease in children compared to adults has been reported by numerous studies, but the mechanisms underlying this phenomenon have not been characterized. It is still unclear how frequently children get infected, and/or generate immune responses to SARS-CoV-2. We have performed immune profiling of pediatric and adult COVID-19 patients in Brazil, producing over 38 thousand data points, asking if cellular or humoral immune responses could help explain milder disease in children. In this study, pediatric COVID-19 patients presented high viral titers. Though their non-specific immune profile was dominated by naive, non-activated lymphocytes, their dendritic cells expressed high levels of HLA-DR and were low in CX3CR1, indicating competence to generate immune responses that are not targeted to inflamed tissue. Finally, children formed strong specific antibody and T cell responses for viral structural proteins. Children’s T cell responses differed from adults in that their CD8+ TNFα+ T cell responses were low for S peptide but significantly higher against N and M peptide pools. Altogether, our data support a scenario in which SARS-CoV-2 infected children may contribute to transmission, though generating strong and differential responses to the virus that might associate with protection in pediatric COVID-19 presentation.

Monitoring of Cytomegalovirus-Specific CD4+ and CD8+ T Cell Responses by Cytokine Flow Cytometry in Renal Transplant Recipients

2016 ◽  
Vol 50 (2) ◽  
pp. 224-235 ◽  
Author(s):  
Hafize KILINÇKAYA DOĞAN ◽  
Esvet MUTLU ◽  
Sadi KÖKSOY ◽  
Vural T. YILMAZ ◽  
Hüseyin KOÇAK ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Renal Transplant ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Renal Transplant Recipients ◽  
Transplant Recipients ◽  
Cell Responses ◽  
Cytokine Flow Cytometry

scholarly journals CARD9 Deficiency Increases Hippocampal Injury Following Acute Neurotropic Picornavirus Infection but Does Not Affect Pathogen Elimination

2021 ◽  
Vol 22 (13) ◽  
pp. 6982
Author(s):  
Suvarin Pavasutthipaisit ◽  
Melanie Stoff ◽  
Tim Ebbecke ◽  
Malgorzata Ciurkiewicz ◽  
Sabine Mayer-Lambertz ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Virus Infection ◽  
T Cell Responses ◽  
Neurotropic Virus ◽  
Genetic Ablation ◽  
Cell Responses ◽  
Neuronal Precursor Cells ◽  
The Impact ◽  
The Brain

Neurotropic viruses target the brain and contribute to neurologic diseases. Caspase recruitment domain containing family member 9 (CARD9) controls protective immunity in a variety of infectious disorders. To investigate the effect of CARD9 in neurotropic virus infection, CARD9−/− and corresponding C57BL/6 wild-type control mice were infected with Theiler’s murine encephalomyelitis virus (TMEV). Brain tissue was analyzed by histology, immunohistochemistry and molecular analyses, and spleens by flow cytometry. To determine the impact of CARD9 deficiency on T cell responses in vitro, antigen presentation assays were utilized. Genetic ablation of CARD9 enhanced early pro-inflammatory cytokine responses and accelerated infiltration of T and B cells in the brain, together with a transient increase in TMEV-infected cells in the hippocampus. CARD9−/− mice showed an increased loss of neuronal nuclear protein+ mature neurons and doublecortin+ neuronal precursor cells and an increase in β-amyloid precursor protein+ damaged axons in the hippocampus. No effect of CARD9 deficiency was found on the initiation of CD8+ T cell responses by flow cytometry and co-culture experiments using virus-exposed dendritic cells or microglia-enriched glial cell mixtures, respectively. The present study indicates that CARD9 is dispensable for the initiation of early antiviral responses and TMEV elimination but may contribute to the modulation of neuroinflammation, thereby reducing hippocampal injury following neurotropic virus infection.

scholarly journals Strong specific anti-viral responses in pediatric COVID-19 patients in South Brazil

2021 ◽  
Author(s):  
Tiago Fazolo ◽  
Karina Lima ◽  
Julia Fontoura ◽  
Priscila de Souza ◽  
Gabriel Hilario ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
T Cell Responses ◽  
Humoral Immune ◽  
Cell Responses ◽  
Humoral Immune Responses ◽  
Hla Dr ◽  
Data Points ◽  
Viral Responses ◽  
Immune Profiling

Abstract Epidemiological evidence that COVID-19 manifests as a milder disease in children compared to adults has been reported by numerous studies, but the mechanisms underlying this phenomenon have not been characterized. It is still unclear how frequently children get infected, and/or generate immune responses to SARS-CoV-2. We have performed immune profiling of pediatric and adult COVID-19 patients in Brazil, producing over 38 thousand data points, asking if cellular or humoral immune responses could help explain milder disease in children. In this study, pediatric COVID-19 patients presented high viral titers. Though their non-specific immune profile was dominated by naive, non-activated lymphocytes, their dendritic cells expressed high levels of HLA-DR and were low in CX3CR1, indicating competence to generate immune responses that are not targeted to inflamed tissue. Finally, children formed strong specific antibody and T cell responses for viral structural proteins. Children’s T cell responses differed from adults in that their CD8+ TNFα+ T cell responses were low for S peptide but significantly higher against N and M peptide pools. Altogether, our data support a scenario in which SARS-CoV-2 infected children may contribute to transmission, though generating strong and differential responses to the virus that might associate with protection in pediatric COVID-19 presentation.

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