Fig. 4. Cytokine production by K8+/+ and K8–/– colon LP cells. (A) Cells were stimulated with PMA/ionomycin and stained for surface CD4 and intracellular cytokines (IL-5, IL-10, TNF, IL-4, IFN or IL-13) or isotype control antibodies. Samples were analyzed by FACS with gating on CD4+ T cells. To confirm specificity of the IL-4 and IL-13 staining, stimulated cells were pre-incubated with excess unlabeled recombinant (r) cytokines (rIL-4 or rIL-13). The frequency of cytokine-producing CD4+ T cells is indicated in the quadrants as percentages (e.g. 1% for IL-5 and 1.8% for IL-10 for K8+/+ CD4+ T cells). (B) Summary of the result shown in A. CD4+ T cells from K8–/– colons produced higher Th2 cytokines (IL-5, IL-4, and IL-13), and IL-10. Data is presented as mean±s.e.m. (n=4). (C) Cytokine production by K8+/+ and K8–/– colon LP cell culture. Cells were cultured in plates coated with anti-CD3 and soluble anti-CD28 (Stimulated) or in the presence of medium alone (Unstimulated) for 48 hours. Culture supernatants from the stimulated cells were analyzed by ELISA for IFN, TNF, IL-10, IL-5 and IL-4 production. Note that K8–/– cultures produced higher levels of Th2 cytokines. Data are presented as mean±s.e.m. (n=3). *P<0.05 when comparing K8–/– with K8+/+.