Supplementary Material for: Dietary Fructo-Oligosaccharides Attenuate Early Activation of CD4+ T Cells Which Produce both Th1 and Th2 Cytokines in the Intestinal Lymphoid Tissues of a Murine Food Allergy Model

<p><b><i>Background:</i></b> Fructo-oligosaccharides (FOS) are prebiotic agents with immunomodulatory effects involving improvement of the intestinal microbiota and metabolome. In this study, we investigated the cellular mechanisms through which FOS modulate intestinal antigen-specific CD4<sup>+</sup> T cell responses in food allergy, using OVA23-3 mice. <b><i>Methods:</i></b> OVA23-3 mice were fed an experimental diet containing either ovalbumin (OVA) or OVA and FOS for 1 week. Body weight and mucosal mast cell protease 1 in the serum were measured as the indicator of intestinal inflammation. Single-cell suspensions were prepared from intestinal and systemic lymphoid tissues for cellular analysis. Cytokine production was measured by ELISA. Activation markers and intracellular cytokines in CD4<sup>+</sup> T cells were analyzed by flow cytometry. Activated CD4<sup>+</sup> T cells were purified to examine cytokine production. <b><i>Results:</i></b> Dietary intake of FOS provided moderate protection from the intestinal inflammation induced by the OVA-containing diet. FOS significantly reduced food allergy-induced Th2 cytokine responses in intestinal tissues but not in systemic tissues. FOS decreased OVA diet-induced IFN-γ<sup>+</sup>IL-4<sup>+</sup> double-positive CD4<sup>+</sup> T cells and early-activated CD45RB<sup>high</sup>CD69<sup>+</sup>CD4<sup>+</sup> T cells in the mesenteric lymph nodes. Furthermore, we confirmed that these CD45RB<sup>high</sup>CD69<sup>+</sup>CD4<sup>+</sup> T cells are able to produce high levels of IFN-γ and moderate level of IL-4, IL-10, and IL-13. <b><i>Conclusions:</i></b> Dietary intake of FOS during the development of food allergy attenuates the induction of intestinal Th2 cytokine responses by regulating early activation of naïve CD4<sup>+</sup> T cells, which produce both Th1 and Th2 cytokines. Our results suggest FOS might be a potential food agent for the prevention of food allergy by modulating oral sensitization to food antigens.</p>