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Switching from ELISpot to FluoroSpot enables the analysis of spot-forming units representing cells producing different cytokines as well as the frequencies of spots derived from cells co-secreting multiple cytokines. Due to the fluorescent read-out signal, sophisticated reader instruments can also measure the relative spot volume, making it possible to differentiate between spots generated by cells secreting different levels of one or more cytokines. Here we describe how triple FluoroSpot assays can be used to define polyfunctional T cells secreting multiple cytokines and how different T-cell populations can differ in the levels of cytokines they secrete., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cytokine ELISPOT has become a powerful routine tool for the analysis of disease- as well as vaccine-induced T-cell responses. The method is limited, however, in that only one cytokine at a time is assessed. Fluorospot is based on the principle of ELISPOT, but facilitates the analysis of single cells secreting several cytokines, e.g., polyfunctional T cells, suggested to be of protective importance in various infectious diseases. By detecting each cytokine with a specific fluorophore and analyzing differentially colored spots by fluorophore--specific filter systems, cells producing single or multiple cytokines are identified. Fluorospot maintains the simplicity and sensitivity of the ELISPOT while taking the analysis a step forward toward multiplex analysis.

Evaluation of cytokine production in macaques has been hampered by a lack of availability of optimized and standardized immunoassays such as ELISA and enzyme-linked immune spot assay (ELISpot); only a limited number of macaque cytokines have been assessed by ELISpot. Using monoclonal antibodies (mAb) to human cytokines that cross-react with cynomolgus and rhesus macaque interferon-gamma (IFN-gamma), interleukin (IL)-2, IL-4, IL-5, IL-6, IL-12, IL-13 and granulocyte monocyte colony-stimulating factor, we measured macaque cytokine production by ELISA and ELISpot. Quantitation of spontaneous as well as phytohemagglutinin (PHA)-induced cytokine production in peripheral blood mononuclear cells (PBMC) from rhesus and cynomolgus macaques and humans were compared. The proportional distribution of the different cytokines, in terms of PBMC synthesizing different cytokines as well as the levels of the different cytokines produced, were similar in all species. Spontaneous- and PHA-induced cytokine productions thus appear to be similarly regulated in macaques and man. ELISpot and ELISA assays for macaque IFN-gamma were further used to measure antigen-specific immune responses of PBMC from cynomolgus macaques exposed to, or vaccinated against, simian immunodeficiency virus (SIV). The establishment of reliable immunoassays for detection of macaque cytokines is of importance for future progress of research utilizing macaques as experimental animals.

Introduction: Contact allergy to nickel (Ni) is a delayed-type hypersensitivity reaction mediated by Ni-reactive T cells producing the hallmark cytokines of several T-helper cell (Th) populations including IFN-γ (Th1), IL-4, IL-5 and IL-13 (Th2), and IL-17A (Th17). IL-22-expressing CD4+ cells, which could be either Th17 co-expressing IL-22 or Th22, expressing IL-22 in the absence of IL-17A, have also been found in Ni-provoked skin of allergic subjects. It has been unclear if Ni-reactive T cells consist of distinct Th cell type populations or if they secrete a mix of Th cell hallmark cytokines. The aim herein was to assess if cellular cytokine responses to Ni, in ex vivo-stimulated peripheral blood mononuclear cells (PBMCs) from Ni-allergic subjects, include not only Th1, Th2, and Th17 but also Th22 hallmark cytokines and to define if the cytokines are produced by distinct cell populations representing different Th profiles. Methods: PBMC from Ni-allergic subjects (n = 15) with different degrees of patch test reactivity and non-allergic controls (n = 5) were in vitro stimulated with Ni. Cytokine levels in PBMC supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA) (IFN-γ, IL-2, IL-3, IL-5, IL-6, IL-13, IL-17A, IL-22, and IL-31). FluoroSpot was used to assess if individual Ni-reactive cells produced single, or combinations of, cytokines representing different Th profiles. Cytokine combinations analyzed were IL-17A/IL-22/IFN-γ, IL-5/IL-17A/IFN-γ, IL-13/IL-22/IFN-γ, and IL-5/IL-13. Results: IL-22 as well as all other cytokines measured by ELISA were induced by Ni at higher levels in PBMC from allergic versus non-allergic subjects, with higher levels being associated with stronger patch test reactivity. The levels of most Ni-induced cytokines were positively correlated with each other; IL-2 displayed the highest correlation with other cytokines and IL-6 the lowest. FluoroSpot analysis showed that Th signature cytokines, IFN-γ (Th1), IL-5 and IL-13 (Th2), IL-17A (Th17), and IL-22 (Th22), were almost exclusively produced by distinct cell populations. Conclusion: Distinct Th cell populations, including Ni-reactive cells displaying Th1, Th2, Th17, and Th22 cytokine profiles, are all increased in PBMC from Ni-allergic subjects and positively associated with patch test reactivity. The relevance of these different Th profile populations for the up- or down-regulation of inflammatory reactions in the skin of Ni-allergic subjects remains to be clarified. [ABSTRACT FROM AUTHOR]

The involvement of T-helper (Th)1, Th17 and Th22 cell subsets, in immunity, as well as in pathological inflammatory reactions, makes it important to determine their relative proportion. A triple FluoroSpot detecting the hallmark cytokines of Th1 (IFN-γ), Th17 (IL-17A) and Th22 (IL-22) was developed and evaluated using human peripheral blood mononuclear cells from healthy donors incubated with tetanus toxoid, Candida albicans extract, mycobacterial purified protein derivative or medium only. Antigen stimulation yielded mainly cells secreting IFN-γ, IL-17A or IL-22 alone but lower proportions of doublesecreting cells were also found; triple-secreting cells were rare. The response to C. albicans contrasted in that higher proportions of IL-17A single secreting as well as co-secreting cells, in particular IL-17A/IL-22, were found. The FluoroSpot analysis correlated well with single cytokine ELISpot assays ran in parallel and the methods displayed a comparable sensitivity. The results demonstrate the functionality of the FluoroSpot assay for simultaneous analysis of distinct Th1, Th17, Th22 as well as intermediate cell populations. The method provides a mean for a simple and rapid analysis of the involvement of these cells in immunity and disease. [ABSTRACT FROM AUTHOR]

Detection of cytokines secreted by ex vivo antigen-stimulated peripheral blood mononuclear cells (PBMC) by ELISA is hampered by low frequencies of specific T cells and cellular receptor consumption. We investigated if ELISpot, measuring cytokine production at the single cell level, facilitated a better detection of the Th2 cytokines IL-4 and IL-5. PBMC from nickel-allergic (n = 31) and non-allergic subjects (n = 10) were stimulated with nickel or tetanus toxoid (TT) and cytokine production assessed by ELISpot and ELISA. By IL-4 ELISpot, 74% of the allergic and 0% control subjects responded to nickel and 56% of all subjects to TT. ELISA detected IL-4 after nickel stimulation only in 13% of the allergic subjects. Also detection of TT-induced IL-5 was improved by ELISpot with 54% subjects responding versus 24% in ELISA. In contrast, detection of nickel-induced IL-5 was more comparable between methods, most likely due to the 7-fold higher IL-5 production per cell in response to nickel versus TT. The low IL-5 response to TT was associated with a higher induction of the down-regulatory cytokine IL-10 by TT as compared to nickel (p < 0.001). Overall, ELISpot displayed a better detection of IL-4 as well as low intensity IL-5 responses thus emphasizing the importance of selecting suitable methods for the measurement of cytokine production ex vivo. [ABSTRACT FROM AUTHOR]

Abstract: Human Transforming Growth Factor (TGF)-β1, one of three TGF-β isoforms, is a pleotropic cytokine critical for many physiological and immunological processes. TGF-β1 is secreted in a latent form, linked to Latency Associated Protein (LAP). Analysis of Latent TGF-β1 by TGF-β1 ELISA requires dissociation of TGF-β1 from LAP, e.g. by acidification of samples. The ELISA then measures total TGF-β1, equivalent to dissociated Latent TGF-β1 plus any free TGF-β1 present prior to acidification. Evolutionary conservation of TGF-β1 across mammals also renders TGF-β1 ELISAs reactive with TGF-β1 in bovine serum often used in human cell cultures. To enable a direct analysis of Latent TGF-β1, monoclonal antibodies were made against LAP from human Latent TGF-β1 and used to develop a LAP ELISA detecting Latent TGF-β1. The ELISA did not react with LAP from human Latent TGF-β2 or 3, respectively, nor with Latent TGF-β in bovine serum. EDTA-containing plasma from healthy subjects (n=20) was analyzed by conventional TGF-β1 ELISA and LAP ELISA. By TGF-β1 ELISA, total TGF-β1 were detected in all samples (median 133pM, range 34–348pM); low levels of free TGF-β1 found in 8/20 non-acidified samples showed that >98.5% of the total TGF-β1 derived from Latent TGF-β1. Latent TGF-β1 found in non-acidified samples by LAP ELISA (median 154pM, range 48–403pM) was comparable in molar levels to, and correlated with, total TGF-β1 (rs 0.96, p<0.0001). A similar agreement between the total TGF-β1 and the LAP ELISA was found with citrate- and heparin-containing plasma. The LAP ELISA facilitates analysis of Latent TGF-β1 without sample acidification and is not compromised by the presence of bovine serum in human cell supernatants. [Copyright &y& Elsevier]

Abstract: Interleukin-4 (IL-4) contains three structurally important intramolecular disulfides that are required for the bioactivity of the cytokine. We show that the cell surface of HeLa cells and endotoxin-activated monocytes can reduce IL-4 intramolecular disulfides in the extracellular space and inhibit binding of IL-4 to the IL-4Rα receptor. IL-4 disulfides were in vitro reduced by thioredoxin 1 (Trx1) and protein disulfide isomerase (PDI). Reduction of IL-4 disulfides by the cell surface of HeLa cells was inhibited by auranofin, an inhibitor of thioredoxin reductase that is an electron donor to both Trx1 and PDI. Both Trx1 and PDI have been shown to be located at the cell surface and our data suggests that these enzymes are involved in catalyzing reduction of IL-4 disulfides. The pro-drug N-acetylcysteine (NAC) that promotes T-helper type 1 responses was also shown to mediate the reduction of IL-4 disulfides. Our data provides evidence for a novel redox dependent pathway for regulation of cytokine activity by extracellular reduction of intramolecular disulfides at the cell surface by members of the thioredoxin enzyme family. [Copyright &y& Elsevier]