Improved Detection of Cytokines Produced by Invariant NKT Cells

www.nature.com/scientificreports OPEN Improved Detection of Cytokines Produced by Invariant NKT Cells Duygu Sag1,2, Müge Özkan3, Mitchell Kronenberg Received: 23 August 2017 Accepted: 16 November 2017 Published: xx xx xxxx 4,5 & Gerhard Wingender 1,4 Invariant Natural killer T (iNKT) cells rapidly produce copious amounts of multiple cytokines after in vivo activation, allowing for the direct detection of a number of cytokines directly ex vivo. However, for some cytokines this approach is suboptimal. Here, we report technical variations that allow the improved detection of IL-4, IL-10, IL-13 and IL-17A ex vivo. Furthermore, we describe an alternative approach for stimulation of iNKT cells in vitro that allows a significantly improved detection of cytokines produced by iNKT cells. Together, these protocols allow the detection of iNKT cell cytokines ex vivo and in vitro with increased sensitivity. Invariant natural killer T (iNKT) cells are innate-like T cells that share features with natural killer (NK) cells and memory T lymphocytes. Antigens for iNKT cells are largely glycolipids, like the model antigen α-galactosylceramide (αGalCer), which are presented by CD1d, a non-polymorphic MHC class I homolog. Following antigenic activation, subsets of iNKT cells rapidly produce copious amounts of cytokines, including Th1, Th2 and Th17 cytokines as well as IL-101–6. However, the detection of some of these cytokines via intracellular cytokine staining (ICCS) is often poor, which is especially the case after in vitro activation of primary iNKT cells. This has led to inconsistencies in the published data for the production of several iNKT cell cytokines and hampers the use of in vitro assays to study the functions of primary iNKT cells. We present here several enhancements to purification and staining protocols that allow improved detection of cytokines, including GM-CSF, IFNγ, IL-2, IL-4, IL-10, IL-13, IL-17A and TNF, produced by primary iNKT cells, ex vivo or in vitro. Results Influence of the fixation method on cytokine detection. Following activation with αGalCer in vivo the majority of iNKT cells produced IL-4, which can be detected directly ex vivo, meaning without the need for TCR cross-linking or pharmacologic activators, and without a requirement for culture in the presence of blockers of protein transport through the Golgi apparatus (Fig. 1A). However, the intensity of the staining tended to be low (Fig. 1A and data not shown) and this can at times make the discrimination of positive events difficult. Therefore, we tested several alternatives for staining and fixation to improve the intracellular staining for IL-4, combined with the use of different fluorophores. We found that fixation of the cells with Cytofix/Cytoperm for 10 minutes at 37 °C, instead of the recommended 4 °C, also significantly increased the staining intensity for most IL-4 conjugates, without negatively affecting surface staining (Fig. 1A and data not shown). This was seen with both of the αIL-4-antibody clones, 11B11 and BVD6-24G2, that were tested (Fig. 1A). The increased staining intensity allowed significantly more iNKT cells to be detected as IL-4+ in the case of FITC- and PE-Cy7-conjugated antibodies, but not in the case of AF647- and PE-CF594-conjugated antibodies (Fig. 1A and Supplementary Figure 1A). A similar variability in the percent of the activated iNKT cells classified as cytokine positive also was noted for IL-2 and IL-13 staining. This depended on the antibody conjugates tested, and for some of the conjugates, fixation at 37 °C led to an increased staining intensity (Fig. 1B). In contrast, no difference in the staining intensity of other cytokine tested, namely GM-CSF, IFNγ, IL-10, IL-17A and TNF, was observed (Fig. 1B, Supplementary Figure 1B, and data not shown). Importantly, changing the temperature of the fixation step did not negatively affecting the surface staining of any of the tested markers (Supplementary Figure 2). Therefore, fixation of activated iNKT cells at 37 °C instead of 4 °C is preferable for IL-2, IL-4 and IL-13 detection. 1 Izmir Biomedicine and Genome Center (IBG), 35340, Balcova, Izmir, Turkey. 2Department of Medical Biology, Faculty of Medicine, Dokuz Eylul University, 35340, Balcova, Izmir, Turkey. 3Izmir International Biomedicine and Genome Institute (IBG-Izmir), Dokuz Eylul University, 35340, Balcova, Izmir, Turkey. 4La Jolla Institute for Allergy and Immunology (LJI), 9420 Athena Circle, La Jolla, CA, 92037, USA. 5Division of Biological Sciences, University of California San Diego, La Jolla, CA, 92037, USA. Correspondence and requests for materials should be addressed to G.W. (email: ) Scientific REPOrTS | 7: 16607 | DOI:10.1038/s41598-017-16832-1 1 www.nature.com/scientificreports/ Figure 1. Fixation method influenced the detection in iNKT cells of several cytokines. (A–C) C57BL/6 mice were either mock treated or injected i.v. with 1μg αGalCer and 90 min later the expression of the cytokines IL-4 (clone BVD6-24G2 or 11B11, as indicated in the histogram) (A), IL-2 (JES6-5H4) (B) and IL-13 (13A) (C) by splenic iNKT cells was analyzed by intracellular cytokine staining (ICCS). Cells were fixed with Cytofix/ Cytoperm for 10 min at either 4 °C or 37 °C as indicated. A summary graph (left) and representative data from gated iNKT cells (right) are in adjacent panels. The fluorochromes conjugated to the antibodies utilized are indicated below the histograms. ns = not statistically significant. Representative data from one of at least three independent experiments are shown. iNKT cell IL-17A requires in vitro cytokine accumulation. In recent years, functional subsets of iNKT cells have been defined7,8. The definition of iNKT cell subsets is largely based on their expression of transcription factors and their function, especially significant biases in cytokine production of the respective iNKT cell types. NKT19, NKT29,10 and NKT1711–14 cells are defined as the iNKT cell subsets biased towards Th1, Th2 or Th17 cytokines, respectively. The underlying gene programs are imprinted during thymic development15. NKT10 cells were characterized by IL-10 production16–20. NKTFH21–23 and FoxP3+ iNKT24,25 cells were defined based on their similarities with TFH and FoxP3+ T cells, respectively. However, the detection of IL-10 and IL-17A production by activated iNKT cells of the appropriate functional subtype is particularly poor when the cells were analyzed directly ex vivo (Figs 2A, 3A and data not shown). For the detection of cytokines produced by conventional, MHC class II-reactive T cells, an in vitro incubation of the cells after purification in the presence of Golgi-transport inhibitors is routinely used to improve cytokine detection26. We adopted this method for the detection of IL-17A production by iNKT cells. Mice were injection i.v. with αGalCer and 90 min later splenocytes were obtained and cultured for 2 h in the presence of Golgi-transport inhibitors (...truncated)