IL-4 in situ staining

International Immunology, 1999

IL-4 is a cytokine which can be expressed by a number of cell types including T h 2 cells, mast cells and a population of CD4 ⍣ NK1.1 ⍣ NK T cells. Although phenotypic markers exist for identifying each of these cell types, there is at present no known cell surface marker common to all IL-4-producing cells. Using gene targeting in embryonic stem cells, we have modified the IL-4 locus by knock-in of a transmembrane domain to generate mice that express a membrane-bound form of IL-4 (mIL-4). Flow cytometry using an IL-4-specific mAb allowed the detection of IL-4-secreting T h 2 cells, mast cells and NK T cells from mIL-4 mice. Furthermore, the analysis of immune responses in mIL-4 mice following immunization with anti-CD3 and anti-IgD has allowed us to identify distinct subpopulations of IL-4-producing NK T cells. Thus, the expression of IL-4 in a membrane-bound form provides a novel method for the identification and characterization of IL-4-producing cells.

Journal of Immunological Methods, 1993

Interleukin-4 (IL-4) is an important T cell and mast cell product that participates in allergic and cytotoxic responses, as well as functions as a growth factor for B, T, and inflammatory cells. Studies of the expression of IL-4 by T cells present in inflammatory reactions would be facilitated by using polymerase chain reaction (PCR) coupled to reverse transcription of mRNA to amplify the small quantity of mRNA present in these cells. In order to use this method in a quantitative manner, a plasmid was constructed that contained a modified form of mouse IL-4 cDNA. This plasmid was transcribed to produce cRNA for this modified sequence. The cRNA was used as an internal standard for the reverse transcription and amplification of IL-4 transcripts in RNA samples from mouse thymocytes. Amplification of reverse-transcribed native IL-4 mRNA produced a 286 bp PCR product. Amplification of the reverse-transcribed standard RNA produced a 155 bp product, which reflected a deletion introduced into the original IL-4 cDNA sequence. Comparison of the amount of the 286 bp native product to the amount of 155 bp standard product enabled the quantitative determination of IL-4 expression in each sample. This method was used to demonstrate that platelet activating factor increases the expression of IL-4 in mouse thymocytes and in a mouse T cell line. The expression of IL-4 by thymocytes exposed to platelet-activating factor (PAF) may reveal an important link between inflammation and the maturation of T cells in the thymus.

Proceedings of The National Academy of Sciences, 1988

We have made use of RNA\cdot RNA in situ hybridization to study the presence of cells producing mRNA for interleukin 4 (IL-4) in the developing thymus, spleen, and T-cell line 2.19. Approximately 1 of 300-400 spleen cells expressed detectable IL-4 mRNA 24 hr after their stimulation by the lectin concanavalin A. Spleen cells were also induced to express mRNA for

Cellular Immunology, 1991

Cytometry, 2001

Comparative Immunology, Microbiology and Infectious Diseases, 2004

We produced four monoclonal antibodies (mAb) and two polyclonal antibodies using the purified cytokine expressed in bacteria and characterized them. Specific binding of each of the mAb and polyclonal antibodies to recombinant swine IL-4 (rSwIL-4) purified from Escherichia coli and baculovirus was demonstrated in an indirect ELISA and/or in western blotting. We established a sandwich enzyme-linked immunosorbent assay (ELISA) for measuring concentration of SwIL-4 in biological samples and established an enzyme-linked immunospot (ELISPOT) assay for detecting IL-4-secreting cells using a mAb and a polyclonal IgG from goat. The detection limit of the sandwich ELISA for SwIL-4 was 78 pg/ml. Using sandwich ELISA, SwIL-4 was detected in the bronchoalveolar lavage fluid (BALF) of pigs experimentally infected with Mycoplasma hyopneumoniae and could quantitate in supernatants of mitogen-stimulated PBMC culture. The ELISPOT system is useful for the detection of IL-4 producing cells in swine PBMC culture.

Frontiers in Immunology, 2022

Despite the knowledge that cell-mediated immunity (CMI) contributes to the reduction of severe influenza infection, transmission, and disease outcome, the correlates of protection for cell-mediated immunity remain still unclear. Therefore, measuring the magnitude and quality of influenza-specific T cell responses in a harmonized way is of utmost importance to improve characterisation of vaccine-induced immunity across different clinical trials. The present study, conducted as part of the FLUCOP project, describes the development of a consensus protocol for the intracellular cytokine staining (ICS) assay, in order to reduce inter-laboratory variability, and its qualification. In order to develop a consensus protocol, the study was divided into different stages. Firstly, two pilot studies evaluated critical parameters in the analytical (read-outs) and post-analytical (gating strategies and data analysis) methods applied by eight different laboratories within the FLUCOP consortium. The methods were then harmonized by fixing the critical parameters and the subsequent consensus protocol was then qualified by one FLUCOP member. The antigen-specific cell population was defined as polypositive CD4 + T cells (i.e. positive for at least two markers among CD40L/IFNg/IL2/TNFa), which was shown to be the most sensitive and specific read-out. The qualification of this consensus protocol showed that the quantification of polypositive CD4 + T cells was precise, linear and accurate, and sensitive with a lower limit of quantification of 0.0335% antigen-specific polypositive CD4 + T cells. In conclusion, we provide the description of a harmonized ICS assay, which Frontiers in Immunology frontiersin.org 01

European Journal of Immunology, 1992

Over the past five years, Massive Open Online Courses (MOOCs) have a remarkable ability to expand access to a large scale of participants worldwide to attend free online courses, beyond the formality of the higher education systems. MOOCs have unique features that support a movement toward a vision of lifelong and on-demand learning for those who are working full time or have taken a break from formal education. Despite their popularity and the large scale participation, a variety of concerns and criticism in the use of MOOCs have been raised. The original concept of MOOCs that aims at breaking down obstacles to education for anyone, anywhere and at any time is far away from the reality. In fact, most MOOC implementations so far still follow a top-down, controlled, teacher-centered, and centralized learning model. Endeavors to implement bottom-up, student-centered, truly open, decentralized, and distributed forms of MOOCs are exceptions rather than the rule. Moreover, the lack of human interaction is the major limitation of the existing MOOCs. Other limitations of MOOCs include pedagogical problems concerning assessment and feedback, the lack of interactivity around the video content, as well as the complexity and diversity of MOOC participants. Furthermore, a major problem with MOOCs is the ignorance of the importance and benefits of face-toface communication. These limitations raise some serious concerns on what role MOOCs should play, or how they should fit into the higher education landscape as an alternative model of teaching and learning and a substantial supplement.

Historia, Vol. 46-47 , pp. 63-102 , 2021