scholarly journals Development of a Simple and Robust Whole Blood Assay with Dual Co-Stimulation to Quantify the Release of T-Cellular Signature Cytokines in Response to Aspergillus fumigatus Antigens

2021 ◽  
Vol 7 (6) ◽  
pp. 462
Author(s):  
Chris D. Lauruschkat ◽  
Lukas Page ◽  
P. Lewis White ◽  
Sonja Etter ◽  
Helen E. Davies ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Whole Blood ◽  
Mononuclear Cells ◽  
T Helper Cell ◽  
Helper Cell ◽  
Medical Mycology ◽  
Enzyme Linked Immunosorbent Assay ◽  
T Helper ◽  
High Discriminatory Power ◽  
Cost Efficient

Deeper understanding of mold-induced cytokine signatures could promote advances in the diagnosis and treatment of invasive mycoses and mold-associated hypersensitivity syndromes. Currently, most T-cellular immunoassays in medical mycology require the isolation of mononuclear cells and have limited robustness and practicability, hampering their broader applicability in clinical practice. Therefore, we developed a simple, cost-efficient whole blood (WB) assay with dual α-CD28 and α-CD49d co-stimulation to quantify cytokine secretion in response to Aspergillus fumigatus antigens. Dual co-stimulation strongly enhanced A. fumigatus-induced release of T-cellular signature cytokines detectable by enzyme-linked immunosorbent assay (ELISA) or a multiplex cytokine assay. Furthermore, T-cell-dependent activation and cytokine response of innate immune cells was captured by the assay. The protocol consistently showed little technical variation and high robustness to pre-analytic delays of up to 8 h. Stimulation with an A. fumigatus lysate elicited at least 7-fold greater median concentrations of key T-helper cell signature cytokines, including IL-17 and the type 2 T-helper cell cytokines IL-4 and IL-5 in WB samples from patients with Aspergillus-associated lung pathologies versus patients with non-mold-related lung diseases, suggesting high discriminatory power of the assay. These results position WB-ELISA with dual co-stimulation as a simple, accurate, and robust immunoassay for translational applications, encouraging further evaluation as a platform to monitor host immunity to opportunistic pathogens.

scholarly journals T helper cell polarisation as a measure of the maturation of the immune response

2003 ◽  
Vol 12 (5) ◽  
pp. 285-292 ◽  
Author(s):  
Scott B. Cameron ◽  
Ellen H. Stolte ◽  
Anthony W. Chow ◽  
Huub F. J. Savelkoul
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Intracellular Cytokine Staining ◽  
T Helper Cell ◽  
Helper Cell ◽  
Enzyme Linked Immunosorbent Assay ◽  
Intracellular Cytokine ◽  
T Helper

Background:T helper cell polarisation is important under chronic immune stimulatory conditions and drives the type of the evolving immune response. Mice treated with superantigensin vivodisplay strong effects on Thsubset differentiation. The aim of the study was to detect the intrinsic capacity of T cells to polarise under variousex vivoconditions.Methods:Purified CD4+T cells obtained from superantigen-treated mice were cultured under Thpolarising conditionsin vitro. By combining intracellular cytokine staining and subsequent flow cytometric analysis with quantitative cytokine measurements in culture supernatants by enzyme-linked immunosorbent assay (ELISA), the differential Thpolarising capacity of the treatment can be detected in a qualitative and quantitative manner.Results and conclusions:BALB/c mice were shown to be biased to develop strong Th2 polarised immune responses using Th0 stimulation of purified CD4+T cells from phosphate-buffered saline-treated mice. Nevertheless, our analysis methodology convincingly showed that even in these mice, Toxic Shock Syndrome Toxin-1 treatmentin vivoresulted in a significantly stronger Th1 polarising effect than control treatment. Our results indicate that populations of Thcells can be assessed individually for their differential Th1 or Th2 maturation capacityin vivoby analysing robustin vitropolarisation cultures combined with intracellular cytokine staining and ELISA.

Defective T-helper cell function after T-cell–depleting therapy affecting naive and memory populations

Blood ◽  
2002 ◽  
Vol 99 (11) ◽  
pp. 4053-4062 ◽  
Author(s):  
Andreas Heitger ◽  
Patricia Winklehner ◽  
Petra Obexer ◽  
Johannes Eder ◽  
Claudia Zelle-Rieser ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Mononuclear Cells ◽  
Cell Function ◽  
Interleukin 2 ◽  
T Helper Cell ◽  
Helper Cell ◽  
High Dose ◽  
T Helper

Impaired T-cell function after T-cell– depleting (TCD) therapy has been hypothesized to be related to a transient predominance of extrathymically expanding memory T cells. To test whether after TCD therapy the naive T-helper cell population is functionally intact, the in vitro immune response of CD4+CD45RA+ (naive) and of CD4+CD45RA− (memory) cells to polyclonal mitogens (immobilized anti-CD3, phytohemagglutinin) was analyzed by flow cytometry in 22 pediatric patients after high-dose chemotherapy (including 5 after autologous and 5 after allogeneic stem cell support). At 1 to 3 months after TCD therapy, patient samples showing decreased lymphoproliferative responses also showed a reduced induction of the early activation marker CD69 by CD4+ T cells from 4 to 72 hours after stimulation even when supplemented with exogenous interleukin-2. This defect affected CD4+CD45RA− cells, but, strikingly, also CD4+CD45RA+ cells, including samples in which CD4+CD45RA+ cells were more than 90/μL, thus indicating ongoing thymopoiesis. Histogram analyses showed the median peak channel of CD69 in control CD4+CD45RA+cells rising 98-fold (median) but only 28-fold in patient cells (P < .0001). Apoptosis as detected by annexin V staining was increased in resting patient CD4+ T cells (25% versus 6%) and also affected CD4+CD45RA+ cells (12% versus 5%, P < .01). When peripheral blood mononuclear cells (PBMCs) were enriched for T cells, stimulatory responses of CD4+ cells and of CD4+CD45RA+ cells markedly improved. Thus, after TCD therapy suppressor factors contained in the non–T-cell fraction of PBMCs may affect T-helper cells irrespective of their naive or memory phenotype thus extending T-cell dysfunction to the presumably thymus-dependently regenerated T cells.

scholarly journals Broad Specificity of Virus-Specific CD4+ T-Helper-Cell Responses in Resolved Hepatitis C Virus Infection

2002 ◽  
Vol 76 (24) ◽  
pp. 12584-12595 ◽  
Author(s):  
Cheryl L. Day ◽  
Georg M. Lauer ◽  
Gregory K. Robbins ◽  
Barbara McGovern ◽  
Alysse G. Wurcel ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mononuclear Cells ◽  
Hcv Infection ◽  
T Helper Cell ◽  
Helper Cell ◽  
T Helper ◽  
Cell Responses ◽  
Chronic Hcv Infection ◽  
Chronic Hcv

ABSTRACT Vigorous virus-specific CD4+ T-helper-cell responses are associated with successful control of hepatitis C virus (HCV) and other human viral infections, but the breadth and specificity of responses associated with viral containment have not been defined. To address this we evaluated the HCV-specific CD4+ T-helper-cell response in HCV antibody-positive persons who lack detectable plasma viremia, and compared this response to that in persons with chronic HCV infection. Peripheral blood mononuclear cells were stimulated with HCV proteins, followed by measurement of HCV-specific CD4+-T-cell responses to a comprehensive set of overlapping HCV peptides by intracellular gamma interferon production. In three persons with resolved HCV infection studied in detail, 13 to 14 epitopes were targeted, but none was recognized by all three. The 37 defined epitopes were predominantly distributed among the HCV proteins core, NS3, NS4, and NS5. In an expanded analysis of responses to these proteins in persons with resolved infection, an average of 10 epitopes was targeted, whereas in persons with chronic viremia never was more than one epitope targeted (P < 0.001). This comprehensive analysis of the breadth and specificity of HCV-specific T-helper-cell responses indicates that up to 14 viral epitopes can be simultaneously targeted by circulating virus-specific CD4+ T helper cells in a controlled human viral infection. Moreover, these data provide important parameters for evaluation of candidate HCV vaccines, and provide rationale for immunotherapy in chronic HCV infection.

Clinical-scale isolation of the total Aspergillus fumigatus–reactive T–helper cell repertoire for adoptive transfer

Cytotherapy ◽  
2015 ◽  
Vol 17 (10) ◽  
pp. 1396-1405 ◽  
Author(s):  
Petra Bacher ◽  
Andrea Jochheim-Richter ◽  
Nadine Mockel-Tenbrink ◽  
Olaf Kniemeyer ◽  
Eva Wingenfeld ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Adoptive Transfer ◽  
T Helper Cell ◽  
Helper Cell ◽  
Clinical Scale ◽  
T Helper ◽  
Cell Repertoire

scholarly journals Nonredundant role of CCRL2 in lung dendritic cell trafficking

Blood ◽  
2010 ◽  
Vol 116 (16) ◽  
pp. 2942-2949 ◽  
Author(s):  
Karel Otero ◽  
Annunciata Vecchi ◽  
Emilio Hirsch ◽  
Jennifer Kearley ◽  
William Vermi ◽  
...  
Keyword(s):  
Dendritic Cell ◽  
Mononuclear Cells ◽  
Inflammatory Responses ◽  
T Helper Cell ◽  
Interleukin 4 ◽  
Helper Cell ◽  
Cell Trafficking ◽  
T Helper

Abstract Chemokine CC motif receptor-like 2 (CCRL2) is a heptahelic transmembrane receptor that shows the highest degree of homology with CCR1, an inflammatory chemokine receptor. CCRL2 mRNA was rapidly (30 minutes) and transiently (2-4 hours) regulated during dendritic cell (DC) maturation. Protein expression paralleled RNA regulation. In vivo, CCRL2 was expressed by activated DC and macrophages, but not by eosinophils and T cells. CCRL2−/− mice showed normal recruitment of circulating DC into the lung, but a defective trafficking of antigen-loaded lung DC to mediastinal lymph nodes. This defect was associated to a reduction in lymph node cellularity and reduced priming of T helper cell 2 response. CCRL2−/− mice were protected in a model of ovalbumin-induced airway inflammation, with reduced leukocyte recruitment in the BAL (eosinophils and mononuclear cells) and reduced production of the T helper cell 2 cytokines, interleukin-4 and -5, and chemokines CCL11 and CCL17. The central role of CCRL2 deficiency in DC was supported by the fact that adoptive transfer of CCRL2−/− antigen-loaded DC in wild-type animals recapitulated the phenotype observed in knockout mice. These data show a nonredundant role of CCRL2 in lung DC trafficking and propose a role for this receptor in the control of excessive airway inflammatory responses.

Expansion of HCV-specific T cells with a follicular T helper cell phenotype after DAA mediated antigen removal

2018 ◽  
Vol 56 (01) ◽  
pp. E2-E89
Author(s):  
M Smits ◽  
C Fauvelle ◽  
T Baumert ◽  
C Neumann-Haefelin ◽  
R Thimme ◽  
...  
Keyword(s):  
T Cells ◽  
T Helper Cell ◽  
Helper Cell ◽  
Cell Phenotype ◽  
T Helper ◽  
Antigen Removal
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